Exosomes from clonal progenitor cells

ABSTRACT

The invention provides methods, compositions, uses and kits relating to exosomes isolated from progenitor cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/020,869, filed on Jul. 3, 2014, the entire contents of which arehereby incorporated by reference.

FIELD OF THE INVENTION

The field of the invention relates to exosomes isolated from progenitorcells.

BACKGROUND

Exosomes are believed to contain important signaling molecules that mayprovide the source of trophic factors responsible for some regenerativebenefits seen in cell replacement therapy. As such they would provide analternative to some cell based therapies that would be easier tomanufacture on a large scale and potentially safer to administer to asubject in need of cell therapy. In particular, the risk associated withtransmission of infectious agents such as viruses may be lower comparedto transplanting whole cells. Moreover, the risk of immune rejection ofthe exosomes relative to transplanted cells may also be lower.Accordingly, exosomes may provide an attractive alternative or adjunctto cell based therapies and cell based regenerative medicine.

Exosomes are 30 to 120 nm vesicles secreted by a wide range of mammaliancell types. Keller et al. (2006) Immunol Lett. 107(2):102; Camussi etal. (2010) Kidney International 78:838. The vesicles are enclosed by alipid bilayer and are larger than LDL which has a size of 22 nm, butsmaller than a red blood cell, which is 6000 to 8000 nm in diameter andhas a thickness of 2000 nm Keller et al. (2006) Immunol Lett.107(2):102.

Exosomes are found both in cells growing in vitro as well as in vivo.They can be isolated from tissue culture media as well as bodily fluidssuch as plasma, urine, milk and cerebrospinal fluid. George et al.(1982) Blood 60:834; Martinez et al. (2005) Am J Physiol Health CirPhysiol 288:H1004. Exosomes originate from the endosomal membranecompartment. They are stored in intraluminal vesicles withinmultivesicular bodies of the late endosome. Multivesicular bodies arederived from the early endosome compartment and contain within themsmaller vesicular bodies that include exosomes. Exosomes are releasedfrom the cell when multivesicular bodies fuse with the plasma membrane.Methods of isolating exosomes from cells has been described, see e.g. USPatent Application Publication No. 20120093885

Exosomes contain a variety of molecules including proteins, lipids andnucleic acids such as DNA, mRNA and miRNA. Their contents are believedto play a part in cell to cell communication involving the release ofthe exosome from one cell and the binding/fusion of the exosome with asecond cell, wherein the contents of the exosomal compartment arereleased within the second cell.

It has been reported that exosomes derived from endothelial progenitorcells may act as vehicle for mRNA transport among cells. These exosomeswere shown to incorporate into normal endothelial cells by interactingwith the α4β1 integrin. Once incorporated into the endothelial cells,the exosomes stimulated an angiogenic program. Deregibus et al. (2007)Blood 110:2440. Similar results were obtained in vivo using severecombined immunodeficient mice. Exosome stimulated endothelial cellsimplanted subcutaneously in Matrigel (a murine sarcoma extract)organized into a patent vessel network connected with the murinevasculature. Deregibus, supra. Bruno et al. (2009) J Am Soc Nephrol20:1053; Herrera et al. (2010) J Cell Mol Med 14:1605.

Of the various molecular cargo of exosomes, miRNAs have recentlyattracted a lot of attention due to their regulatory roles in geneexpression. MiRNAs are small, non-coding regulatory RNAs that can have awide range of effects on multiple RNA targets, thus having the potentialto have greater phenotypic influence than coding RNAs. MiRNA profiles ofexosomes often differ from those of the parent cells. Profiling studieshave demonstrated that miRNAs are not randomly incorporated intoexosomes but rather a subset of miRNAs is preferentially packaged intoexosomes, suggesting an active sorting mechanism of exosomal miRNAs.Guduric-Fuchs et al. (2014) Nucleic Acid Res. 42:9195; Ohshima et al.(2010) PloS One 5(10):e13247.

Because exosomes contain a variety of molecules, many believed to playan important role in cell signaling, exosomes would prove useful inresearch and industry and would have applications as therapeutics,diagnostics and in screening assays. Frequently, however, theavailability of reproducible, essentially identical populations ofexosomes is limited by the fact that most sources of exosomes are cellsthat senesce and thus have limited replicative capacity. Accordingly,there is a need for exosomes that are derived from a clonal source thathas an extended replicative capacity that is greater than most adult orfetal derived cells. The invention described infra meets this need andas well as other needs in the field.

SUMMARY OF THE INVENTION

In various embodiments described herein the invention providescompositions comprising exosomes obtained from progenitor cell lines, aswell as methods of making and using exosomes obtained from progenitorcell lines.

The isolation of embryonic progenitor cells has been described. See Westet al. (2008) Regen Med 3:287; US Patent Application Publication Nos.20080070303 20100184033. Embryonic progenitors are cell lines derivedunder a variety of culture conditions from pluripotent stem cells, suchas human embryonic stem (hES) cells or induced pluripotent stem (iPS)cells. The progenitor cell lines are clonal and while they do, in mostinstances, senesce, they also possess longer telomeres compared to adultor fetal derived tissue or cells (such as adult stem cells) andaccordingly have enhanced replicative capacity relative to those celltypes. Because of their clonality and their enhanced replicativecapacity they provide a suitable source of exosomes that will offer thebenefit of uniformity with regard to the exosome composition andabundance relative to exosomes derived from their typical sources suchas adult cells or adult stem cells.

In certain embodiments the invention provides an exosome isolated from aprogenitor cell line, such as clonal progenitor cell line.

In certain embodiments the invention provides an exosome isolated from ahuman progenitor cell line, such as a clonal human progenitor cell line.

In some embodiments the invention provides an exosome isolated fromendothelial progenitor cell.

In some embodiments the invention provides an exosome isolated from aclonal human endothelial progenitor cell.

In other embodiments the invention provides an exosome isolated from the30-MV2-6 human clonal progenitor cell line.

In further embodiments the invention provides an exosome isolated from ahuman clonal progenitor cell that expresses CD31 and CD34.

In certain embodiments the invention provides an exosome isolated from ahuman progenitor cell line, wherein the human progenitor cell is not anadult stem cell.

In further embodiments the invention provides an exosome isolated from ahuman progenitor cell line, wherein the human progenitor cell is not amesenchymal stem cell (MSC).

In certain embodiments the invention provides an exosome isolated from acell that has not been transfected with an exogenous gene.

In certain other embodiments the invention provides an exosome isolatedfrom a cell that has been transfected with an exogenous gene, whereinthe gene is not c-myc.

In yet other embodiments the invention provides an exosome isolated froma cell that does not overexpress c-myc.

In other embodiments the invention provides an exosome isolated from the30-MV2-6 clonal human progenitor cell line.

In still other embodiments the invention provides an exosome isolatedfrom a cell expressing one or more genes chosen from the genes listed inTable 1.

In further embodiments the invention provides an exosome isolated from acell expressing a plurality of the genes chosen from the genes listed inTable 1.

In yet other embodiments the invention provides an exosome isolated froma cell expressing the genes listed in Table 1.

In some embodiments, the invention provides an exosome containing CD63.

In other embodiments, the invention provides an exosome containing oneor more miRNAs listed in Table 2 or Table 4.

In further embodiments, the invention provides an exosome containing oneor more angiogenic miRNAs.

In yet further embodiments, the invention provides an exosome containingmiR-126.

In some embodiments the invention provides an exosome isolated from ahuman clonal progenitor cell, wherein the exosome contains one or moremiRNAs listed in Table 2 or Table 4.

In other embodiments the invention provides an exosome isolated from ahuman clonal progenitor cell, wherein the exosome contains one or moreangiogenic miRNAs.

In yet other embodiments the invention provides an exosome isolated froma human clonal progenitor cell, wherein the exosome contains miR-126.

In further embodiments the invention provides an exosome isolated from ahuman clonal progenitor cell, wherein the exosome contains CD63.

In some embodiments the invention provides an exosome that induces acell to form vascular tube like structures.

In other embodiments the invention provides an exosome that induces acell to form branching vascular tube like structures.

In yet other embodiments the invention provides a cell culturecomprising an exosome isolated from a progenitor cell and a cell whichwas not the source of the isolated exosome.

In certain embodiments the invention provides a cell culture comprisingan exosome isolated from a progenitor cell and a cell which was not thesource of the isolated exosome, wherein the cell has the ability to formvascular tube like structures.

In further embodiments the invention provides a cell culture comprisingan exosome isolated from a progenitor cell and a cell which was not thesource of the isolated exosome, wherein the cell is an endothelial cell.

In still further embodiments the invention provides a cell culturecomprising an exosome isolated from a progenitor cell and a cell whichwas not the source of the isolated exosome, wherein the cell is a humanumbilical vein endothelial cell (HUVEC).

In the cell culture embodiments described above the progenitor cell maybe a human progenitor cell, such as a human embryonic progenitor cell.One example of a human embryonic progenitor cell is the 30-MV2-6 cellline.

In the cell culture embodiments described above the progenitor cell maybe, for example, a clonal progenitor cell line, an oligoprogenitor cellline. The progenitor cell may express one or more genes listed inTable 1. The progenitor cell may express a plurality of the genes listedin Table 1. The progenitor cell line may express the genes listed inTable 1. The progenitor cell line may express CD31 and CD34.

In some embodiments the invention provides a method of isolating anexosome from a progenitor cell, such as a clonal progenitor cellcomprising 1) culturing the progenitor cell in a suitable media orbuffer for a time sufficient to allow the cells to exocytose exosomesinto the culture media; 2) harvesting the media from the cell culture ofstep 1; and 3) isolating the exosomes from the media of step 2, therebyisolating an exosome from a clonal progenitor cell.

In some embodiments the invention provides a method of isolating anexosome from a human clonal progenitor cell comprising 1) culturing thehuman clonal progenitor cell in a suitable media or buffer for a timesufficient to allow the human clonal progenitor cell to exocytoseexosomes into the culture media; 2) harvesting the media from the cellculture of step 1; and 3) isolating the exosomes from the media of step2, thereby isolating an exosome from a clonal progenitor cell.

In other embodiments the invention provides a method of isolating anexosome from a 30-MV2-6 human clonal progenitor cell line comprising 1)culturing the 30-MV2-6 human clonal progenitor cell line in a suitablemedia or buffer for a time sufficient to allow the 30-MV2-6 human clonalprogenitor cell line to exocytose exosomes into the culture media; 2)harvesting the media from the cell culture of step 1; and 3) isolatingthe exosomes from the media of step 2, thereby isolating an exosome froma 30-MV2-6 human clonal progenitor cell line.

In still other embodiments the invention provides a method of isolatingan exosome from a human clonal progenitor cell line expressing CD31 andCD34 comprising 1) culturing the human clonal progenitor cell lineexpressing CD31 and CD34 in a suitable media or buffer for a timesufficient to allow the human clonal progenitor cell line expressingCD31 and CD34 to exocytose exosomes into the culture media; 2)harvesting the media from the cell culture of step 1; and 3) isolatingthe exosomes from the media of step 2, thereby isolating an exosome froma human clonal progenitor cell line expressing CD31 and CD34.

In some embodiments the invention provides a method of isolating anexosome from a human clonal progenitor cell line expressing one or moreof the genes listed in Table 1 comprising 1) culturing the human clonalprogenitor cell line expressing one or more of the genes listed in Table1 in a suitable media or buffer for a time sufficient to allow the humanclonal progenitor cell line expressing one or more of the genes listedin Table 1 to exocytose exosomes into the culture media; 2) harvestingthe media from the cell culture of step 1; and 3) isolating the exosomesfrom the media of step 2, thereby isolating an exosome from a humanclonal progenitor cell line expressing one or more of the genes listedin Table 1.

In still other embodiments the invention provides a method of isolatingan exosome from a human clonal progenitor cell line expressing aplurality of the genes listed in Table 1 comprising 1) culturing thehuman clonal progenitor cell line expressing a plurality of the geneslisted in Table 1 in a suitable media or buffer for a time sufficient toallow the human clonal progenitor cell line expressing a plurality ofthe genes listed in Table 1 to exocytose exosomes into the culturemedia; 2) harvesting the media from the cell culture of step 1; and 3)isolating the exosomes from the media of step 2, thereby isolating anexosome from a human clonal progenitor cell line expressing a pluralityof the genes listed in Table 1.

In further embodiments the invention provides a method of isolating anexosome from a human clonal progenitor cell line expressing the geneslisted in Table 1 comprising 1) culturing the human clonal progenitorcell line expressing the genes listed in Table 1 in a suitable media orbuffer for a time sufficient to allow the human clonal progenitor cellline expressing the genes listed in Table 1 to exocytose exosomes intothe culture media; 2) harvesting the media from the cell culture of step1; and 3) isolating the exosomes from the media of step 2, therebyisolating an exosome from a human clonal progenitor cell line expressingthe genes listed in Table 1.

In some embodiments the invention provides a method of isolating anexosome from a human clonal progenitor cell wherein the human clonalprogenitor cell line has not been transfected with an exogenous genecomprising 1) culturing the human clonal progenitor cell line that hasnot been transfected with an exogenous gene in a suitable media orbuffer for a time sufficient to allow the human clonal progenitor cellline that has not been transfected with an exogenous gene to exocytoseexosomes into the culture media; 2) harvesting the media from the cellculture of step 1; and 3) isolating the exosomes from the media of step2, thereby isolating an exosome from a human clonal progenitor cell linethat has not been transfected with an exogenous gene.

In other embodiments the invention provides a method of isolating anexosome from a human clonal progenitor cell, wherein the human clonalprogenitor cell line has been transfected with an exogenous gene,wherein the gene is not c-myc, comprising 1) culturing the human clonalprogenitor cell line that has been transfected with an exogenous gene,wherein the exogenous gene is not c-myc, in a suitable media or bufferfor a time sufficient to allow the human clonal progenitor cell linethat has been transfected with an exogenous gene, wherein the exogenousgene is not c-myc, to exocytose exosomes into the culture media; 2)harvesting the media from the cell culture of step 1; and 3) isolatingthe exosomes from the media of step 2, thereby isolating an exosome froma human clonal progenitor cell line that has been transfected with anexogenous gene, wherein the exogenous gene is not c-myc.

In still other embodiments the invention provides a method of isolatingan exosome from a human clonal progenitor cell wherein the human clonalprogenitor cell line does not overexpress c-myc comprising 1) culturingthe human clonal progenitor cell line that does not overexpress c-myc ina suitable media or buffer for a time sufficient to allow the humanclonal progenitor cell line that has not been transfected with anexogenous gene to exocytose exosomes into the culture media; 2)harvesting the media from the cell culture of step 1; and 3) isolatingthe exosomes from the media of step 2, thereby isolating an exosome froma human clonal progenitor cell line that does not overexpress c-myc.

In further embodiments the invention provides a method of inducing orenhancing a cells ability to form vascular tube like structurescomprising contacting a cell capable of making vascular tube likestructures with an exosome isolated from a progenitor cell therebyinducing or enhancing a cells ability to form vascular tube likestructures.

In still further embodiments the invention provides a method of inducingor enhancing a cells ability to form vascular tube like structurescomprising contacting an endothelial cell with an exosome isolated froma progenitor cell thereby inducing or enhancing an endothelial cellsability to form vascular tube like structures.

In other embodiments the invention provides a method of inducing orenhancing a cells ability to form vascular tube like structurescomprising contacting a cell capable of making vascular tube likestructures with an exosome isolated from a clonal progenitor cellthereby inducing or enhancing a cells ability to form vascular tube likestructures.

In further embodiments the invention provides a method of inducing orenhancing a cells ability to form vascular tube like structurescomprising contacting a cell capable of making vascular tube likestructures with an exosome isolated from a human clonal progenitor cellthereby inducing or enhancing a cells ability to form vascular tube likestructures.

In certain embodiments the invention provides a method of inducing orenhancing a cells ability to form vascular tube like structurescomprising contacting a cell capable of making vascular tube likestructures with an exosome isolated from a human endothelial progenitorcell thereby inducing or enhancing a cells ability to form vascular tubelike structures.

In yet other embodiments the invention provides a method of inducing orenhancing a cells ability to form vascular tube like structurescomprising contacting a cell capable of making vascular tube likestructures with an exosome isolated from a human clonal endothelialprogenitor cell thereby inducing or enhancing a cells ability to formvascular tube like structures.

In further embodiments the invention provides a method of inducing orenhancing a cells ability to form vascular tube like structurescomprising contacting a cell capable of making vascular tube likestructures with an exosome isolated from a cell expressing one or moregenes listed in Table 1 thereby inducing or enhancing a cells ability toform vascular tube like structures.

In other embodiments the invention provides a method of inducing orenhancing a cells ability to form vascular tube like structurescomprising contacting a cell capable of making vascular tube likestructures with an exosome isolated from a cell expressing a pluralityof genes listed in Table 1 thereby inducing or enhancing a cells abilityto form vascular tube like structures.

In yet other embodiments the invention provides a method of inducing orenhancing a cells ability to form vascular tube like structurescomprising contacting a cell capable of making vascular tube likestructures with an exosome isolated from a cell expressing the geneslisted in Table 1 thereby inducing or enhancing a cells ability to formvascular tube like structures.

In still other embodiments the invention provides a method of inducingor enhancing a cells ability to form vascular tube like structurescomprising contacting a cell capable of making vascular tube likestructures with an exosome isolated from a cell expressing the markersCD31 and CD34 thereby inducing or enhancing a cells ability to formvascular tube like structures.

In further embodiments the invention provides a method of inducing orenhancing a cells ability to form vascular tube like structurescomprising contacting a cell capable of making vascular tube likestructures with an exosome isolated from a 30-MV2-6 cell therebyinducing or enhancing a cells ability to form vascular tube likestructures.

In still other embodiments the invention provides a method ofregeneration a tissue or an organ comprising contacting one or morecells capable of regenerating a tissue or an organ with an exosomeisolated from a progenitor cell.

In yet other embodiments the invention provides a method of regeneratinga vascular tissue or organ comprising contacting a cell capable ofregenerating a vascular tissue or organ with an exosome isolated from aprogenitor cell.

In some embodiments the invention provides a method of regenerating avascular tissue or organ comprising contacting a cell capable ofregenerating a vascular tissue or organ with an exosome isolated from ahuman clonal endothelial progenitor cell.

In further embodiments the invention provides a method of regenerating avascular tissue or organ comprising contacting a cell capable ofregenerating a vascular tissue or organ with an exosome isolated from a30-MV2-6 cell.

In certain embodiments the invention provides a method of treating asubject in need of vascular therapy comprising administering an exosomeisolated from a progenitor cell.

In some embodiments the invention provides a method of treating asubject in need of vascular therapy comprising administering an exosomeisolated from a human clonal progenitor cell.

In further embodiments the invention provides a method of treating asubject in need of vascular therapy comprising administering an exosomeisolated from an endothelial progenitor cell.

In certain embodiments the invention provides a method of treating asubject in need of vascular therapy comprising administering an exosomeisolated from human clonal endothelial progenitor cell.

In yet other embodiments the invention provides a method of treating asubject in need of vascular therapy comprising administering an exosomeisolated from a 30-MV2-6 human endothelial progenitor cell.

In still other embodiments the invention provides a method of treating asubject in need of vascular therapy comprising administering an exosomeisolated from a human clonal progenitor cell expressing CD31 and CD34.

In further embodiments the invention provides a method of treating asubject in need of vascular therapy comprising administering an exosomeisolated from a human clonal progenitor cell expressing one or moregenes listed in Table 1.

In some embodiments the invention provides a method of treating asubject in need of vascular therapy comprising administering an exosomeisolated from a human clonal progenitor cell expressing a plurality ofgenes listed in Table 1.

In yet other embodiments the invention provides a method of treating asubject in need of vascular therapy comprising administering an exosomeisolated from a human clonal progenitor cell expressing the markerslisted in Table 1.

In further embodiments the invention provides a kit comprising anexosome isolated from a progenitor cell and at least one container.

BRIEF DESCRIPTION OF DRAWINGS

For a fuller understanding of the nature and advantages of the presentinvention, reference should be had to the following detailed descriptiontaken in connection with the accompanying drawings, in which:

FIG. 1 shows a graph of the size and concentration of exosomes isolatedfrom a) human embryonic progenitor cell line 30-MV2-6; and b) the HT1080cell line.

FIG. 2A shows three photomicrographs, the first showing the effects onvascular tube like formation in HUVECs grown in the presence of basalmedia supplemented with exosomes isolated from human embryonicprogenitor cell line 30-MV2-6 (top); the second showing the effects onvascular tube formation in HUVECs grown in base media supplemented withPBS, but without exposure to exosomes isolated from human embryonicprogenitor cell line 30-MV-2-6 (middle); and the third showing theeffects on vascular tube like formation in HUVECs grown in completemedium, but without exposure to exosomes isolated from human embryonicprogenitor cell line 30-MV2-6 (bottom).

FIG. 2B is a graph quantifying four parameters: cell covered area; totaltube length; total number of branching points and total number of loopsin HUVECs grown in the presence of exosomes isolated from humanembryonic progenitor cell line 30-MV2-6 (“MV2-6 EXO”); HUVECs grown inbasal media +PBS, but without exposure to exosomes isolated from humanembryonic progenitor cell line 30-MV2-6 (“Basal PBS”); and HUVECs grownin complete media, but without exposure to exosomes isolated from humanembryonic progenitor cell line 30-MV2-6 (“Complete”).

FIG. 3A is photomicrograph showing that hES derived perivascular cellsform aggregates when cultured in the presence of complete EGM-MV2 mediawith serum and growth factors.

FIG. 3B is a photomicrograph showing that hES derived perivascular cellsform incomplete tubes when cultured in EGM-MV2 basal media.

FIGS. 3C-E are photomicrographs showing that increasing doses ofexosomes isolated from the human clonal endothelial progenitor cell line30-MV2-6 resulted in increasing tube formation in hES derivedperivascular cells.

FIG. 3F is a graph showing the cell covered area of hES derivedperivascular cells grown either in complete media (complete); basalmedia (base) or basal media supplemented with increasing doses (2.5×10⁷,5.0×10⁷, 10.0×10⁷) of exosomes isolated from the human clonalendothelial progenitor cell line 30-MV2-6.

FIG. 3G is a graph showing the total number of branching points in hESderived perivascular cells grown either in complete media (complete);basal media (base) or basal media supplemented with increasing doses(2.5×10⁷, 5.0×10⁷, 10.0×10⁷) of exosomes isolated from the human clonalendothelial progenitor cell line 30-MV2-6.

FIG. 3H is a graph showing the total tube length of vascular tube likestructures formed by hES derived perivascular cells grown either incomplete media (complete); basal media (base) or basal mediasupplemented with increasing doses (2.5×10⁷, 5.0×10⁷, 10.0×10⁷) ofexosomes isolated from the human clonal endothelial progenitor cell line30-MV2-6.

FIG. 31 is a graph showing the total number of loops formed by hESderived perivascular cells grown either in complete media (complete);basal media (base) or basal media supplement with increasing doses(2.5×10⁷, 5.0×10⁷, 10.0×10⁷) of exosomes isolated from the human clonalendothelial progenitor cell line 30-MV2-6.

FIG. 4 shows comparison of in vitro angiogenic activity of exosomesisolated from the human clonal endothelial cell line 30-MV2-6 versusexosomes isolated from bone marrow mesenchymal stem cells (BM-MSC). Twodifferent commercially available sources of BM-MSCs were used, Promocell(panel A) and Lonza (panel B). 30-MV2-6: exosomes derived from 30-MV2-6cell line; MSC: exosomes derived from BM-MSC; BM: basal medium, negativecontrol; CM: complete growth medium. Results were normalized to tubelength obtained using complete growth medium (CM).

FIG. 5 shows that the angiogenic activity of exosomes isolated from the30-MV2-6 cell line is dose dependent (panel A) and at least six timesmore potent than the angiogenic activity of exosomes isolated fromBM-MSCs (panel B).

FIG. 6A depicts analysis of miRNA content in 30-MV2-6 derived exosomesversus BM-MSC derived exosomes. 30-MV2-6 exosome RNA was compared toBM-MSC RNA for miRNA content using an 84 miRNA PCR array. The scatterplot indicates miRNAs with greater than 6-fold differences. The uppercircled dot represents miRNA miR-126-3p, which is expressed at 77.6-foldhigher in 30-MV2-6 exosomes than in BM-MSC exosomes. The lower circleddot represents miRNA miR-376c-3p which is expressed at 752-fold lower in30-MV2-6 exosomes than in MB-MSC exosomes.

FIG. 6B is a bar graph illustrating the differences (measured asfold-change) in miRNA expression profile between 30-MV2-6-derivedexosomes versus BM-MSC-derived exosomes.

FIG. 7 consists of 5 photomicrographs showing in vivo angiogenicactivity of human clonal endothelial cell line 30-MV2-6 in the Matrigelplug assay in immunocompromised mice. Blood vessel-like structures areseen in the exosome treated plugs (panels A and C) but not in thecontrol plug (panel E). Endothelial cell content is confirmed bystaining with anti-Von Willebrand factor antibody (panels B and D).

FIG. 8 shows that the in vitro angiogenic activity of exosomes derivedfrom 30-MV2-6 cells grown in T-flasks is comparable to the in vitroangiogenic activity of exosomes derived from 30-MV2-6 cells grown in theQuantum cell expansion bioreactor. Panel A depicts tube networkformation and panel B depicts quantitative analysis of tube length usingImage J angiogenesis analyzer software. BM: basal medium, negativecontrol; CM: complete growth medium.

FIG. 9 is a bar graph illustrating the effect of oxygen level andconditioning medium used on the angiogenic activity of exosomes derivedfrom 30-MV2-6 cells. Use of PBS versus basal medium as the conditioningmedium had no significant effect on the angiogenic activity of the30-MV2-6 exosomes. Similarly, no significant effect on the angiogenicactivity was observed in conditioning the cells in 1% versus 5% oxygen.

FIG. 10 shows ELISA detection of CD63 on intact exosomes to determineexosome concentration. The standard curve was prepared using exosomes(from HT1080 cells) of known concentration as determined by nanoparticleanalysis (NTA). Quantitation of exosomes in samples of unknownconcentration was calculated from OD value at 450 nm The assay assumesCD63 content of exosomes derived from various different cells remainsrelatively constant.

FIG. 11 depicts the high proliferative capacity and stable angiogenicexosome production of the clonal human embryonic progenitor cell line30-MV2-6. The 30-MV2-6 cells continue to proliferate in cell culturepast 50 population doublings (panel A). Similarly, exosomes that retaintheir angiogenic activity as measured by the in vitro tube formationassay may be prepared from 30-MV2-6 cells that have been cultured for atleast 50 population doublings (panel B).

FIG. 12 is a heat map of Illumina gene expression array (Illumina,Hayward, CA) data showing that the clonal embryonic epithelialprogenitor cell lines derived from the embryonic stem cell line ESI-017have similar endothelial specific gene expression pattern as variousadult endothelial cells from different sources.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.It is also to be understood that the terminology used in the descriptionis for the purpose of describing the particular versions or embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art. Anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of embodiments of the presentdisclosure.

The invention provides exosomes isolated from clonal progenitor cells,such as human clonal progenitor cells derived from a human pluripotentstem cell. Because the cells are clonal and have enhanced replicativecapacity in vitro, the invention provides a means of producing the sameexosomes over and over again. This provides for a consistent productallowing either the researcher or clinician to alleviate any concernsregarding both the quality and the consistency of the exosomes in anyapplication. Accordingly, the invention also provides methods of makingthe progenitor cells from which the exosomes are derived and methods ofisolating the exosomes from these cells. The invention also contemplatesuses, cell cultures and kits comprising the exosomes all of which aredescribed infra. Definitions

As used herein, the singular forms “a,” “an,” and “the” include pluralreference unless the context clearly dictates otherwise. Thus, forexample, reference to a “therapeutic” is a reference to one or moretherapeutics and equivalents thereof known to those skilled in the art,and so forth.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45% to 55%.

“CD31”, also known as PECAM-1 (platelet endothelial cell adhesionmolecule) is a protein in the immunoglobulin superfamily found on thesurface of platelets, monocytes, neutrophils, and some types of T-cells.CD31 makes up a large portion of endothelial cell intercellularjunctions. CD31 is encoded in humans by the PECAM-1 gene and is commonlyused as a marker for endothelial cells.

“CD34” is a cell surface glycoprotein that functions as a cell-celladhesion factor. The CD34 protein is a member of a family of single-passtransmembrane sialomucin proteins that are expressed on earlyhematopoietic and vascular-associated tissue. CD34 is encoded in humansby the CD34 gene. It is commonly used as a marker for hematopoieticand/or vascular endothelial cells.

As used herein, the term “clonal” refers to a population of cellsobtained by the expansion of a single cell into a population of cellsall derived from that original single cell and not containing othercells. The terms “clonal progenitor cell”, “embryonic clonal progenitorcell”, “clonal progenitor cell line” and “embryonic clonal progenitorcell line” each refer to progenitor cell lines that are derivedclonally, i.e., derived by the expansion of a single cell into apopulation of cells all derived from that original single cell and notcontaining other cells.

The term “embryonic stem cell” as used herein refers to a pluripotentcell that is derived from a blastocysts, such as an in vitro fertilizedblastocyst. Embryonic stem cells include human embryonic stem cells,which are available as established cell lines. The established celllines are available commercially from numerous public cell banks, e.g.WiCell and private corporations, e.g. ESI BIO.

The term “human pluripotent cell” or “human pluripotent stem cell” asused herein refers to a human cell which is capable of differentiatinginto at least one cell type found in or derived from each of the threeprimary germ layers. Some human pluripotent stem cells have the abilityto differentiate into all cells found in or derived from each of thethree primary germ layers. Examples of human pluripotent stem cellsinclude human embryonic stem cells (Thomson (1998) Science 282:1145),human embryonic germ cells (Shamblott et al. (2001) PNAS 98:113 andinduced pluripotent cells (Takahashi et al. (2007) Cell 131:861.

The term “induced pluripotent stem cell” as used herein, refers to apluripotent cell that has been genetically reprogrammed using anytechnique known in the art from an adult somatic cell back to thedevelopmentally less mature pluripotent state.

The term “miRNA,” as used herein, refers to microRNA which includes RNAspecies that are 21-25 nt long and may be single- or double-stranded.MicroRNAs are short, non-coding RNA molecules that have been found inanimals, including humans, and in plants. The term encompasses smallinterfering RNA (siRNA) and small temporal RNA (stRNA), as well as miRNAproper. miRNAs are transcribed as parts of longer RNA molecules andprocessed in the nucleus by the dsRNA ribonuclease Drosha to hairpinstructures 70-100 nucleotides long. These are transported to thecytoplasm where they are digested to 21-23-mers by the dsRNAribonuclease Dicer. Single-stranded miRNAs bind to complementarysequences in mRNA thereby inhibiting translation.

“miR-126” is a human microRNA that is specifically expressed inendothelial cells, throughout capillaries and in larger blood vessels.miR-126 plays a role in angiogenesis by regulating the expression levelsof various genes by pre- and post-transcription mechanisms. As usedherein, the term “miR-126” refers to all of the following: the stem-loopmiR-126, miR-126-3p (3′ arm of the hairpin precursor) and miR-126-5p (5′arm of the hairpin precursor). miRNA naming conventions are described inKozomara and Griffiths-Jones, (2014) Nucleic Acids Res. 42(Databaseissue):D68. The terms “miR-126-3p” and “hsa-miR-126-3p” are also usedinterchangeably throughout this application.

The use of “nucleic acid,” “polynucleotide” or “oligonucleotide” orequivalents herein means at least two nucleotides covalently linkedtogether. In some embodiments, an oligonucleotide is an oligomer of 6,8, 10, 12, 20, 30 or up to 100 nucleotides. In some embodiments, anoligonucleotide is an oligomer of at least 6, 8, 10, 12, 20, 30, 40, 50,60, 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides. A“polynucleotide” or “oligonucleotide” may comprise DNA, RNA, cDNA, PNAor a polymer of nucleotides linked by phosphodiester and/or anyalternate bonds.

The term “peptide,” as used herein, refers to two or more amino acidsjoined by a peptide bond. A peptide can, in some instances, be a portionof a full length protein.

The term “protein” as used herein, refers to a full length protein, i.e.one having all of the amino acids coded for by the mRNA that encodes theparticular protein. Also included in the definition are modifiedproteins where one or more amino acids have been cleaved (e.g. a signalsequence) as a result of the protein being secreted from a cell.

By “pharmaceutically acceptable”, it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The term “pluripotent cell” or “pluripotent stem cell” as used herein,refers to a cell which is capable of differentiating into at least onecell type found in or derived from each of the three primary germlayers. Some pluripotent stem cells have the ability to differentiateinto all cells found in or derived from each of the three primary germlayers.

The term “progenitor cell line” as used herein refers to a line of cellsthat is more differentiated (developed) compared to a pluripotent cell,such as iPS cell or an hES cell, but is not terminally differentiated.Progenitor cells will have enhanced replicative capacity compared to aterminally differentiated cell which typically has senesced. Progenitorcells may also have longer telomere lengths compared to a cell that hasterminally differentiated. Progenitor cell lines, when cultured, may beable double in population size at least 5, at least 10, at least 20, atleast 30, at least 40, at least 50 times. In some instances progenitorcell lines may be able to double in population size 5-400 times, 10-300times, 20-200 times, 30-80 times, 40-60 times. One example of aprogenitor cell line is an embryonic progenitor cell. Embryonicprogenitor cell is obtained from a pluripotent cell such as an iPS cellor a hES as previously described. See West et al. (2008) Regen Med3:287; US Patent Application Publication Nos. 20080070303 20100184033.

The term “subject,” as used herein includes, but is not limited to,humans, non-human primates and non-human vertebrates such as wild,domestic and farm animals including any mammal, such as cats, dogs,cows, sheep, pigs, horses, rabbits, rodents such as mice and rats. Insome embodiments, the term “subject,” refers to a male. In someembodiments, the term “subject,” refers to a female.

The term “suitable media,” as used herein, refers to a solution that canbe used to grow cells in culture. A suitable media may include aformulation of salts and/or buffering reagents. A suitable media mayinclude any or all of the following: salts, sugars, amino acids,proteins, growth factors, cytokines, and hormones, additives such asserum, albumin, antibiotics, insulin, selenium and transferrin. Suitableculture media includes for example commercially available culture mediasuch as DMEM, MEM Stem Pro and the like.

A “therapeutically effective amount” of a composition such as atherapeutic agent described infra, e.g. an exosome, is a predeterminedamount calculated to achieve the desired effect. In some embodiments,the effective amount is a prophylactic amount. In some embodiments, theeffective amount is an amount used to medically treat the disease orcondition. The specific dose of a composition administered according tothis invention to obtain therapeutic and/or prophylactic effects will,of course, be determined by the particular circumstances surrounding thecase, including, for example, the composition administered, the route ofadministration, and the condition being treated. It will be understoodthat the effective amount administered will be determined by thephysician in the light of the relevant circumstances including thecondition to be treated, the choice of composition to be administered,and the chosen route of administration. A therapeutically effectiveamount of composition of this invention is typically an amount such thatwhen it is administered in a physiologically tolerable excipientcomposition, it is sufficient to achieve an effective systemicconcentration or local concentration in the targeted tissue.

The terms “treat,” “treated,” or “treating,” as used herein, can referto both therapeutic treatment or prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) an undesiredphysiological condition, symptom, disorder or disease, or to obtainbeneficial or desired clinical results. In some embodiments, the termmay refer to both treating and preventing. For the purposes of thisdisclosure, beneficial or desired clinical results may include, but arenot limited to one or more of the following: alleviation of symptoms;diminishment of the extent of the condition, disorder or disease;stabilization (i.e., not worsening) of the state of the condition,disorder or disease; delay in onset or slowing of the progression of thecondition, disorder or disease; amelioration of the condition, disorderor disease state; and remission (whether partial or total), whetherdetectable or undetectable, or enhancement or improvement of thecondition, disorder or disease. Treatment includes eliciting aclinically significant response. Treatment also includes prolongingsurvival as compared to expected survival if not receiving treatment.

Exosomes

Exosomes of the invention are double membrane bound vesicles secretedfrom cells of plants and animals, such as mammals including humans,non-human primates, dogs, cats, sheep, cows, pigs, horses, rabbits,mice, rats and guinea pigs to name but a few. Thus exosomes may beisolated from any cell type from any source. In some embodiments of theinvention the exosomes of the invention may be secreted from a humancell, such as a human clonal progenitor cell. In some embodiments theexosomes may be secreted from an endothelial human clonal progenitorcell.

The exosomes may contain one or more markers expressed by their cell oforigin. In some embodiments the exosomes contain CD63.

The exosomes may contain one or more miRNAs. In some embodiments, theexosomes of the invention contain one or more miRNAs chosen from Table 2or 4. In some embodiments, the exosomes of the invention contain one ormore angiogenic miRNAs. In some embodiments, the exosomes of theinvention contain miR-126.

Where the exosomes are derived from a clonal progenitor cell, theexosomes will be of uniform quality and composition. Thus the exosomesisolated from a clonal progenitor cell will not vary as a result ofgenetic variation of the source cell. The molecular composition of thecontents and the bio-physical characteristics of the vesicles will beconsistent and reproducible. Moreover, because of the replicativecapacity of the human embryonic progenitor cells, the invention providesan overabundance of the exosomes of the invention. This is in directcontrast with exosomes obtained from other sources known in the artwhere the paucity of the cell type or the problem of senescence limitsthe availability of a reproducible exosome. Moreover, in certainembodiments the cells giving rise to the exosomes of the invention, areneither transformed nor malignant, thus avoiding any possible concernregarding carcinogenesis of the exosomes.

The exosomes of the invention may have diameter ranging from about 20nm-130 nm; from about 30 nm-120 nm; about 40 nm-110 nm; about 50 nm-100nm; about 85 nm-95 nm. In some embodiments the exosomes of the inventionhave a diameter of about 90 nm In some embodiments the exosomes of theinvention have a diameter of about 88 nm.

The exosomes may be comprised of a lipid bilayer containingtrans-membrane proteins and may contain hydrophilic components withinthe vesicle of the exosome. The contents of the vesicle may be derivedfrom the cytoplasm of the cell or from other vesicle structures withinthe cell, e.g., endosomes. The vesicle may contain nucleic acids, suchas DNA, RNA including mRNA, miRNA as well as proteins and peptides.

The exosomes of the invention may serve as depots for the delivery oftherapeutic molecules of any kind. The exosomes of the invention can beengineered to contain therapeutic molecules such as nucleic acids,proteins, peptides, small molecules such as drugs and the like. Anytechnique known in the art can be used to load the exosomes of theinvention with a desired therapeutic molecule. For example cationiclipids could be used to transfect the exosomes with a desired nucleicacid such as DNA, RNA, include mRNA and miRNA. HIV that protein could beused to transport protein or peptide therapeutics into the exosomes ofthe invention. The therapeutic molecules can be chosen, engineered ordesigned to have any desired therapeutic effect. For example moleculesassociated with enhanced angiogenesis could be loaded into the exosomesof the invention, e.g. VEGF.

The secreted exosomes of the invention can be contacted with a targetcell (e.g. a cell that is not the same as the cell of origin for theexosome) such that the exosome is taken up by the target cell, e.g.endocytosed. Once inside the cell, the contents of the vesicle may bereleased into the cytoplasm where the molecules contained within thevesicle may act as signaling molecules in one or more signaling pathwaysthereby inhibiting or enhancing gene expression. The signaling moleculesmay act at the level of transcription or translation for example. Insome instances, where the vesicles contain RNA, the RNA can betranscribed by the target cell. In some instances where the RNA is amiRNA the miRNA can inhibit gene expression.

Methods of Isolating Exosomes

Exosomes may be isolated from any suitable cell that contains exosomes.Described infra are several exemplary cell and cell types that may beused to implement this method. The method may involve seeding the cellat an appropriate density in a tissue culture vessel and then incubatingthe cells in a suitable media or buffer for a suitable period of time.In some embodiments the cells may be permitted to attach to the culturevessel before the exosomes are isolated. In other embodiments the cellsmay be kept in suspension while the exosomes are isolated. The cells maybe permitted to replicate in culture before the exosomes are isolated.Alternatively, the exosomes may be isolated from the cells that have notreplicated, or replicated minimally (e.g. less than 1 doubling).

To initiate the method the cells are seeded in a tissue culture methodat a suitable cell density. The cell density (cells per unit area) mayrange from about 5 k/cm², about 10 k/cm², about 15 k/cm², about 20k/cm², about 25 k/cm², about 30 k/cm², about 35 k/cm², about 40 k/cm²,about 45 k/cm ², about 50 k/cm², about 55 k/cm², about 60 k/cm², about70 k/cm², about 75 k/cm.² In some embodiments the cell density (cellsper unit area) may range from about 1 k/cm²-100 k/cm², 10 k/cm²-90k/cm², 20 k/cm²-80 k/cm², 30 k/cm²-70 k/cm², 40 k/cm²-60 k/cm²′. In oneembodiment the cells are seeded at a density (cells per unit area) of 40k/cm².

The cells may be seeded in any isotonic solution. In one embodiment asuitable solution may include a suitable buffer. Examples of suitablebuffers may include phosphate buffered saline (PBS), HEPES and the like.In other embodiments the cells may be seeded in any suitable cellculture media, many of which are commercially available. Exemplary mediainclude DMEM, RPMI, MEM, Media 199, HAMS and the like. In one embodimentthe media is EGM-MV2. The media may be supplemented with one or more ofthe following: growth factors, cytokines, hormones, serum, such as fetalcalf serum, serum substitutes such as knock out replacement serum orB27, antibiotics, vitamins and/ or small molecule drugs. In oneembodiment the media is supplemented with a TGFβ inhibitor, e.g.SB43154).

The method may be practiced by placing the cells in a suitableenvironment, such as a cell incubator heated to about 37° C. In someembodiments the cells may be incubated at room temperature. Theincubator may be humidified and have an atmosphere that is about 5% CO₂and about 1% O₂. In some embodiments the CO₂ concentration may rangefrom about 1-20%, 2-10%, 3-5%. In some embodiments the O₂ concentrationmay range from about 1-20%, 2-10%, 3-5%.

The method may be practiced by incubating the cells in the media orbuffer for about 1-72 hours, 1-48 hours, 2-24 hours, 3-18 hours, 4-16hours, 5-10 hours. In some embodiments the cells are incubated for about16 hours.

Incubation of the cells as described above allows for the exocytosis ofthe exosomes by the cells into the isotonic solution. After incubationof the cells in the isotonic solution as described above, the isotonicsolution may be harvested. For example the isotonic solution may bepipetted or decanted into another vessel such as a centrifuge tube. Aprecipitating agent may be added to the isotonic solution at this timeto facilitate the precipitation of the exosomes in the solution.Examples of precipitating agents include a solution that is about 15%polyethylene glycol. Alternatively, a commercially preparedprecipitating agent may be used, e.g., Total Exosome Isolation Reagent(Life Technologies, Carlsbad, Calif.). The cells may then be incubatedfor a suitable time period e.g., 1-48 hours, 2-24 hours, 3-18 hours,4-16 hours, 5-10 hours. In some embodiments the cells are incubated forabout 16 hours. The cells may be incubated at a temperature of about 1°C., 5° C., 10° C., 15° C., 20° C., 25° C., 30° C. In one embodiment thecells are incubate at about 4° C.

After incubating the harvested cell conditioned isotonic solution withthe precipitating reagent described above the harvested cell conditionedisotonic solution may be centrifuged at about 1,000×g, 2,000×g, 4,000×g,6,000x g, 8,000×g; 10,0000×g; 12,000×g, 14,000×g , 16,000×g; 18,000×g.In one embodiment the harvested cell conditioned isotonic solution iscentrifuged at about 10,000×g. The harvested cell conditioned isotonicsolution may be centrifuged at about a temperature of 2° C., 4 ° C., 6°C., 8° C., 10° C., 12° C., 14° C., 16° C., 18° C., 20° C., 22° C., 24°C., 26° C. In one embodiment the harvested cell conditioned isotonicsolution are centrifuged at about a temperature of 4° C.

After centrifugation the isotonic solution is removed and the exosomesare resuspended in a suitable buffer such as PBS. The volume of buffermay be about 0.01 volumes-about 0.09 volumes, about 0.02 volumes toabout 0.08 volumes; about 0.03 volumes to about 0.07 volumes of theprecipitating solution. In one embodiment the harvested cell conditionedisotonic solution is resuspended in PBS at a volume equivalent to about0.01 volumes of the precipitating solution. The harvested exosomes maybe used immediately or frozen and stored, e.g., at −20° C., for lateruse.

Progenitor Cells

In certain embodiments of the invention progenitor cells serve as thesource of the exosomes described infra. The progenitor cell may be fromany animal or plant. For example the exosome may be from a mammal, suchas a human, a non-human primate, a horse, a cow, a sheep, a goat, a pig,a cat, a dog, a rabbit, a guinea pig, a rodent such as a mouse or a rat.Typically a progenitor cell will not have an essentially unlimitedreplicative capacity as typically found in embryonic stem cells, butwill nonetheless have, a result of their longer telomeres, a greaterreplicative capacity compared to adult primary cells or tissues (e.g.primary cells) or adult stem cells.

The progenitor cell may be derived from a pluripotent stem cell, such asan embryonic stem cell or an induced pluripotent stem cell. Theprogenitor cell may be a clonal cell or an oligoclonal cell. Anoligoclonal cell would include a population of cells similar cells, e.g.phenotypically or genetically. The progenitor cell may be a clonal humanembryonic progenitor cell. The progenitor cell may be a clonal humanembryonic endothelial progenitor cell. The progenitor cell may be aclonal embryonic progenitor cell that expresses CD31 and CD36. Theprogenitor cell may be a clonal embryonic progenitor cell expressing oneor more genes listed in Table 1. The progenitor cell may be a clonalembryonic progenitor cell expressing a plurality of the genes listed inTable 1. The progenitor cell may be a clonal embryonic progenitor cellexpressing the genes listed in Table 1.

Where the progenitor cells are clonal cells obtained from pluripotentstem cells they will provide an almost unlimited source of the sameexosomes. This is due to two factors: the genetic identity of theoriginal cellular source material and the enhanced telomere lengthsfound in early progenitors which provide for enhanced replicativecapacity relative to adult tissue or cells or adult stem cells.Moreover, unlike adult stem cells which are typically available in verysmall numbers and are difficult to expand in culture, the clonalembryonic progenitors described infra are available in large numbers andare relatively easy to expand in culture.

In some embodiments the progenitor cell is not an adult stem cell. Insome embodiments of the invention the progenitor cell is not an MSC. Insome embodiments the clonal progenitor cell is not transfected orengineered to express an exogenous gene. In some embodiments the clonalprogenitor cell is not transfected to express an oncogene. In someembodiments the clonal progenitor does not express c-myc. In otherembodiments the clonal progenitor cell is transfected or engineered toexpress an exogenous gene. Examples of suitable exogenous genes includethe catalytic component of human telomerase, e.g. hTERT.

Uses of Exosomes

The exosomes described herein may be used in therapeutic, research anddiagnostic applications. For example the exosomes described infra may beadded to a cell culture to enhance one or more phenotypic traits of thecells. The exosomes of the invention may be added to a cell culture toinhibit one or more phenotypic traits of the cells. The exosomes of theinvention may be added to a cell culture to provide a new phenotypictrait of the cells.

The exosomes of the invention may be added to a culture of endothelialcells to enhance the ability of the cells to form vascular tube likestructures. The exosomes of the invention may be added to any cellhaving the ability to form vascular tube like structures to enhance thecells ability to form tube like structures.

In some embodiments the exosomes of the invention are contacted with acell thereby providing at least one new phenotypic trait to the cell.For example, the exosomes of the invention may confer the ability toform vascular tube like structures to cell lacking the ability to formvascular tube like structures before it was contacted with the exosomesof the invention.

In certain embodiments the exosomes of the invention may be added to aculture of perivascular cells to enhance the ability of the perivascularcells to form vascular tube like structures.

In some embodiments the invention provides a method of increasing thelength of a vascular tube like structure formed by a cell such as anendothelial relative to an endothelial cell that has not been treatedwith the exosomes of the invention comprising contacting the endothelialcell with an exosome isolated from a progenitor cell such as a humanclonal progenitor cell, e.g., 30-MV2-6 cells. In some embodiments theinvention provides a method of increasing the length of a vascular tubelike structure formed by a cell such as a perivascular cell relative toa perivascular cell that has not been treated with the exosomes of theinvention comprising contacting the perivascular cell with an exosomeisolated from a progenitor cell such as a human clonal progenitor cell,e.g., 30-MV2-6 cells. In some embodiments the invention provides amethod of increasing the branching of a vascular tube like structureformed by an endothelial cell relative to an endothelial cell that hasnot been treated with the exosomes of the invention comprisingcontacting the endothelial cell with an exosome isolated from aprogenitor cell such as a human clonal progenitor cell, e.g., 30-MV2-6cells. In some embodiments the invention provides a method of increasingthe branching of a vascular tube like structure formed by a perivascularcell relative to a perivascular cell that has not been treated with theexosomes of the invention comprising contacting the perivascular cellwith an exosome isolated from a progenitor cell such as a human clonalprogenitor cell, e.g., 30-MV2-6 cells. In still other embodiments theinvention provides a method of increasing the number of loops in thevascular tube like structures formed by an endothelial cell relative toan endothelial cell that has not been treated with the exosomes of theinvention comprising contacting the endothelial cell with an exosomeisolated from a progenitor cell such as a human clonal progenitor cell,e.g., 30-MV2-6 cells. In yet other embodiments the invention provides amethod of increasing the number of loops in the vascular tube likestructures formed by a perivascular cell relative to a perivascular cellthat has not been treated with the exosomes of the invention comprisingcontacting the perivascular cell with an exosome isolated from aprogenitor cell such as a human clonal progenitor cell, e.g., 30-MV2-6cells.

The exosomes of the invention may be administered therapeutically to asubject in need of treatment. For example the exosomes of the inventionmay be administered to a subject in need of treatment for any diseaserequiring the enhanced ability to form vascular tube like structures.The exosomes of the invention may be used to treat a subject sufferingfrom cardiovascular disease, heart failure, infarction, chronic wounds,ulcer, clogged vessels or arteries, damaged vessels, stenotic vessels,arteriosclerosis, angina, peripheral vascular disease, Alzheimer'sdisease, ischemia, diabetes, cancer, cell replacement transplant ortherapy, tissue and cell regenerative therapy and Parkinson's disease.The exosomes may be used as depot to deliver therapeutic molecules suchas small molecules, nucleic acids, proteins and peptides.

The exosomes of the invention may be directly administered to a subjectin need of treatment or an in vitro cell culture. Alternatively theexosomes can be provided enclosed within a matrix or scaffold. Suitablematrices or scaffolds may include a matrix or scaffold comprised of oneor more extracellular matrix proteins, e.g. laminin, fibronectin and thelike. Other suitable matrices or scaffolds include Matrigel® which is amurine sarcoma extract. The matrix or scaffold may be a hydrogel. Thehydrogel may be comprised of hylauronate and gelatin (see U.S. Pat. Nos.8,324,184; 7,928,069). In one embodiment the exosomes of the inventionmay be delivered in HyStem (Biotime, Inc., Alameda Calif.).

Using the methods described infra along with routine chromatographictechniques known in the art the exosomes of the invention may be used toisolate one or more nucleic acids, proteins or peptides expressed by aprogenitor cell serving as the source of the exosome. Once isolated, theproteins or peptides isolated from the exosomes of the invention can beused to make antibodies to the isolated proteins or peptides (See Harlowet al. Antibodies: A Lab Manual 2^(nd) Edition; Cold Spring Harbor Press2013).

The exosomes of the invention may be used in drug screening assays. Forexample where the exosomes described infra enhance vascular tubeformation in vitro, the exosomes can be used to screen for drugs thatenhance or inhibit this capability. A cell culture comprising cellshaving the ability to form vascular tube like structures may becontacted with the exosomes of the invention and a drug candidate may beapplied to the same cell culture either before, after or simultaneouslywith the exosomes to determine the effect of the drug the ability of theexosomes to enhance vascular tube formation in the cell culture. Theeffects can be compared to untreated cells and cells treated only withthe exosomes of the invention.

Pharmaceutical Compositions

Modes of administration for a therapeutic (either alone or incombination with other pharmaceuticals) can be, but are not limited to,sublingual, injectable (including short-acting, depot, implant andpellet forms injected subcutaneously or intramuscularly), or by use ofvaginal creams, suppositories, vaginal rings, rectal suppositories,intrauterine devices, and transdermal forms such as patches and creams.

Specific modes of administration will depend on the indication. Theselection of the specific route of administration and the dose regimenis to be adjusted or titrated by the clinician according to methodsknown to the clinician in order to obtain the optimal clinical response.The amount of therapeutic to be administered is that amount which istherapeutically effective. The dosage to be administered will depend onthe characteristics of the subject being treated, e.g., the particularanimal treated, age, weight, health, types of concurrent treatment, ifany, and frequency of treatments, and can be easily determined by one ofskill in the art (e.g., by the clinician).

Pharmaceutical formulations containing the therapeutic of the presentdisclosure and a suitable carrier can be solid dosage forms whichinclude, but are not limited to, tablets, capsules, cachets, pellets,pills, powders and granules; topical dosage forms which include, but arenot limited to, solutions, powders, fluid emulsions, fluid suspensions,semi-solids, ointments, pastes, creams, gels and jellies, and foams; andparenteral dosage forms which include, but are not limited to,solutions, suspensions, emulsions, and dry powder; comprising aneffective amount of a polymer or copolymer of the present disclosure. Itis also known in the art that the active ingredients can be contained insuch formulations with pharmaceutically acceptable diluents, fillers,disintegrants, binders, lubricants, surfactants, hydrophobic vehicles,water soluble vehicles, emulsifiers, buffers, humectants, moisturizers,solubilizers, preservatives and the like. The means and methods foradministration are known in the art and an artisan can refer to variouspharmacologic references for guidance. For example, ModernPharmaceutics, Banker & Rhodes, Marcel Dekker, Inc. (1979); and Goodman& Gilman's The Pharmaceutical Basis of Therapeutics, 6th Edition,MacMillan Publishing Co., New York (1980) can be consulted.

The compositions of the present disclosure can be formulated forparenteral administration by injection, e.g., by bolus injection orcontinuous infusion. The compositions can be administered by continuousinfusion subcutaneously over a period of about 15 minutes to about 24hours. Formulations for injection can be presented in unit dosage form,e.g., in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents.

For oral administration, the compositions can be formulated readily bycombining the therapeutic with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the therapeutic of the inventionto be formulated as tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, suspensions and the like, for oral ingestion by apatient to be treated. Pharmaceutical preparations for oral use can beobtained by adding a solid excipient, optionally grinding the resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients include, but are not limited to, fillers such as sugars,including, but not limited to, lactose, sucrose, mannitol, and sorbitol;cellulose preparations such as, but not limited to, maize starch, wheatstarch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,and polyvinyl pyrrolidone (PVP). If desired, disintegrating agents canbe added, such as, but not limited to, the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores can be provided with suitable coatings. For this purpose,concentrated sugar solutions can be used, which can optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments can be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active therapeutic doses.

Pharmaceutical preparations which can be used orally include, but arenot limited to, push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with filler such as, e.g., lactose, binders such as, e.g.,starches, and/or lubricants such as, e.g., talc or magnesium stearateand, optionally, stabilizers. In soft capsules, the active therapeuticcan be dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycols. In addition,stabilizers can be added. All formulations for oral administrationshould be in dosages suitable for such administration.

For buccal administration, the pharmaceutical compositions can take theform of, e.g., tablets or lozenges formulated in a conventional manner.

For administration by inhalation, the therapeutic for use according tothe present disclosure is conveniently delivered in the form of anaerosol spray presentation from pressurized packs or a nebulizer, withthe use of a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the therapeuticand a suitable powder base such as lactose or starch.

The compositions of the present disclosure can also be formulated inrectal compositions such as suppositories or retention enemas, e.g.,containing conventional suppository bases such as cocoa butter or otherglycerides.

In addition to the formulations described previously, the therapeutic ofthe present disclosure can also be formulated as a depot preparation.Such long acting formulations can be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection.

Depot injections can be administered at about 1 to about 6 months orlonger intervals. Thus, for example, the compositions can be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

In transdermal administration, the compositions of the presentdisclosure, for example, can be applied to a plaster, or can be appliedby transdermal, therapeutic systems that are consequently supplied tothe organism.

Pharmaceutical compositions can include suitable solid or gel phasecarriers or excipients. Examples of such carriers or excipients includebut are not limited to calcium carbonate, calcium phosphate, varioussugars, starches, cellulose derivatives, gelatin, and polymers such as,e.g., polyethylene glycols.

The compositions of the present disclosure can also be administered incombination with other active ingredients, such as, for example,adjuvants, protease inhibitors, or other compatible drugs or compoundswhere such combination is seen to be desirable or advantageous inachieving the desired effects of the methods described herein.

In some embodiments, the disintegrant component comprises one or more ofcroscarmellose sodium, carmellose calcium, crospovidone, alginic acid,sodium alginate, potassium alginate, calcium alginate, an ion exchangeresin, an effervescent system based on food acids and an alkalinecarbonate component, clay, talc, starch, pregelatinized starch, sodiumstarch glycolate, cellulose floc, carboxymethylcellulose,hydroxypropylcellulose, calcium silicate, a metal carbonate, sodiumbicarbonate, calcium citrate, or calcium phosphate.

In some embodiments, the diluent component may include one or more ofmannitol, lactose, sucrose, maltodextrin, sorbitol, xylitol, powderedcellulose, microcrystalline cellulose, carboxymethylcellulose,carboxyethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, methylhydroxyethylcellulose, starch, sodiumstarch glycolate, pregelatinized starch, a calcium phosphate, a metalcarbonate, a metal oxide, or a metal aluminosilicate.

In some embodiments, the optional lubricant component, when present,comprises one or more of stearic acid, metallic stearate, sodiumstearylfumarate, fatty acid, fatty alcohol, fatty acid ester,glycerylbehenate, mineral oil, vegetable oil, paraffin, leucine, silica,silicic acid, talc, propylene glycol fatty acid ester, polyethoxylatedcastor oil, polyethylene glycol, polypropylene glycol, polyalkyleneglycol, polyoxyethylene-glycerol fatty ester, polyoxyethylene fattyalcohol ether, polyethoxylated sterol, polyethoxylated castor oil,polyethoxylated vegetable oil, or sodium chloride.

Kits

In some embodiments the invention provides a kit comprising exosomesisolated from a progenitor cell, such as a human clonal progenitor cell.The progenitor cell may be an endothelial progenitor cell, such as humanclonal embryonic progenitor cell, e.g. 30MV2-6. The exosomes may beprovided in one or more containers. The exosomes may be provided in asuitable buffer, e.g. PBS or a suitable media, such as a commerciallyavailable cell culture media, e.g. DMEM. The kit may further contain acell having the ability to form vascular tube like structures. The cellmay be an endothelial cell, e.g. HUVEC and/or a perivascular cell. Thecells may be provided in a suitable media, e.g. DMEM or the like oralternatively the cells may be provided in a buffer such as PBS. In someembodiments the cells may be provided frozen in a suitable freezingmedia such as a commercially available media supplemented with DMSO. Thekit may optionally include instructions as to how to reconstitute theexosomes, culture the cells and/or contact the cells with exosomes so asto enhance vascular tube like formation.

In other embodiments the invention provides a kit comprising a humanclonal embryonic progenitor cell, such as 30-MV2-6. The cell may beprovided in at least one container in suitable media or buffer. The kitmay include buffers and/or media for isolating exosomes from the cells.The kit may contain one or more vessels, e.g. a multi-well plate forculturing the cells. The kit may further contain a cell line capable offorming vascular tube like structures such as endothelial cells.Suitable cells include endothelial cells such as HUVEC and/or a aperivascular cell. Any or all of the cells may be provided frozen in asuitable media, e.g. freezing media such as a commercially availablemedia supplemented with DMSO. The kit may optionally includeinstructions as to how to culture the cells and/or contact theendothelial cells with exosomes isolated from the progenitor cells so asto enhance or induce vascular tube like formation.

Additional Embodiments of the Invention

1. An exosome isolated from a progenitor cell line.

2. The exosome of 1, wherein the progenitor cell line is a humanprogenitor cell line.

3. The exosome of 1, wherein the progenitor cell line is a clonalprogenitor cell line.

4. The exosome of 1, wherein the progenitor cell line is an endothelialprogenitor cell line.

5. The exosome of 1, wherein the exosome contains CD63.

6. The exosome of 1, wherein the exosome contains one or more miRNAslisted in Table 2 or Table 4.

7. The exosome of 1, wherein the exosome contains one or more angiogenicmiRNAs.

8. The exosome of 1, wherein the exosome contains miR-126.

9. The exosome of 1, wherein the progenitor cell line expresses CD31 andCD34.

10. The exosome of 1, wherein the progenitor cell line expresses one ormore genes listed in Table 1.

11. The exosome of 1, wherein the progenitor cell line is 30-MV2-6.

12. The exosome of 1, wherein the exosome enhances the formation ofvascular tube like formations when contacted with an endothelial cell.

13. The exosome of 1 further comprising a pharmaceutical carrier.

14. A method of isolating an exosome from a clonal progenitor cellcomprising 1) culturing the clonal progenitor cell in a suitable mediaor buffer for a time sufficient to allow the clonal progenitor cell toexocytose exosomes into the culture media or buffer; 2) harvesting themedia or buffer from the cell culture of step 1; and 3) isolating theexosomes from the media or buffer of step 2, thereby isolating anexosome from a clonal progenitor cell.

15. The method of 14, wherein the suitable media or buffer is PBS.

16. The method of 14, wherein the suitable media or buffer is EGM-MV2.

17. The method of 14, wherein after step 2 a precipitating agent isadded to the media or buffer.

18. The method of 14, wherein the precipitating agent comprisespolyethylene glycol.

19. The method of 14, wherein step 3 comprises centrifuging theharvested media of buffer.

20. The method of 14, wherein the suitable time of step 1 is about 16hours.

21. The method of 14, wherein after step 2, the method further comprisesa step of incubating harvested media or buffer.

22. The method of 21, wherein the incubation step is performed for about16 hours.

23. The method of 21, wherein the incubation step is performed at about4° C.

24. A cell culture comprising an exosome isolated from a progenitor celland a cell which was not the source of the isolated exosome.

25. The exosome of 24, wherein the progenitor cell line is a humanprogenitor cell line.

26. The exosome of 24, wherein the progenitor cell line is a clonalprogenitor cell line.

27. The exosome of 24, wherein the progenitor cell line is anendothelial progenitor cell line.

28. The exosome of 24, wherein the exosome contains CD63.

29. The exosome of 24, wherein the exosome contains one or more miRNAslisted in Table 2 or Table 4.

30. The exosome of 24, wherein the exosome contains one or moreangiogenic miRNAs.

31. The exosome of 24, wherein the exosome contains miR-126.

32. The exosome of 24, wherein the progenitor cell line expresses CD31and CD34.

33. The exosome of 24, wherein the progenitor cell line expresses one ormore genes listed in Table 1.

34. The exosome of 24, wherein the progenitor cell line is 30-MV2-6.

35. The exosome of 24, wherein the exosome enhances the formation ofvascular tube like formations when contacted with an endothelial cell.

36. A method of inducing or enhancing a cell's ability to form vasculartube like structures comprising contacting a cell capable of makingvascular tube like structures with an exosome isolated from a progenitorcell thereby inducing or enhancing a cells ability to form vascular tubelike structures.

37. The method of 36, wherein the cell capable of making vascular tubelike structures is an endothelial cell.

38. The method of 37, wherein the endothelial cell is a HUVEC.

39. A method of treating a subject in need of vascular therapycomprising administering an exosome isolated from a progenitor cell.

40. The method of 39, wherein the subject is human.

41. The method of 39, wherein the subject exosome is administered to thesubject to treat a condition chosen from cardiovascular disease, heartfailure, infarction, chronic wounds, ulcer, clogged vessels or arteries,damaged vessels, stenotic vessels, arteriosclerosis, angina, peripheralvascular disease, Alzheimer's disease, ischemia, diabetes, cancer, cellreplacement transplant or therapy, tissue and cell regenerative therapyand Parkinson's disease.

42. The method of 39, wherein the progenitor cell line is a humanprogenitor cell line.

43. The method of 39, wherein the progenitor cell line is a clonalprogenitor cell line.

44. The method of 39, wherein the progenitor cell line is an endothelialprogenitor cell line.

45. The method of 39, wherein the exosome contains CD63.

46. The method of 39, wherein the exosome contains one or more miRNAslisted in Table 2 or Table 4.

47. The method of 39, wherein the exosome contains one or moreangiogenic miRNAs.

48. The method of 39, wherein the exosome contains miR-126.

49. The method of 39, wherein the progenitor cell line expresses CD31and CD34.

50. The method of 39, wherein the progenitor cell line expresses one ormore genes listed in Table 1.

51. The method of 39, wherein the progenitor cell line is 30-MV2-6.

52. The method of 39, wherein the exosome enhances the formation ofvascular tube like formations when contacted with an endothelial cell.

53. The method of 39 further comprising a pharmaceutical carrier.

EXAMPLES Example 1 Preparation of Exosomes Derived from a HumanEmbryonic Progenitor Cell Line

Exosomes were prepared from a human embryonic progenitor cell line(PureStem® cell line, ESI Bio, Alameda, Calif.). PureStem® cell linesare scalable clonally pure embryonic progenitor cell lines derived fromhuman embryonic stem (hES) cells (West et al. (2008) Regen Med.3(3):287). The 30-MV2-6 PureStem® cell line is a CD31 positive, CD34positive, endothelial progenitor line derived from the ESI-017 embryonicstem cell line. A gene expression profile of the 30-MV2-6 cells asanalyzed by microarray is provided herein in Table 1, and includes genesyielding relative fluorescence units >1000 rfu.

TABLE 1 30-MV2-6 P6, Gene symbol MBA_3877 EEF1A1 29470.94 EEF1A128581.27 EEF1A1 28032.06 TMSB4X 27627.77 GNB2L1 27295.94 TPT1 26985.77LOC100129758 26697.4 LAIR1 26461.51 F2R 26252 LOC285176 26055.83 RPL4125872.51 NAG18 25689.47 LOC649150 25513.84 FTL 25344.95 LOC9156125177.92 LOC100132593 25023.94 RPLP2 24889.1 LOC388474 24755.51 MGC1670324611.59 UBC 24480.02 LOC389342 24350.84 CLUAP1 24238.08 RPS27 24117.06ZNF674 23992.58 IMAA 23881.17 RRP7B 23769.37 C19orf31 23655.06 ANXA2P223547.03 LOC401206 23440.4 LOC100133876 23333.52 GGA1 23233.85 MSH323140.74 LOC729439 23047.74 LOC440589 22950.21 UBC 22847.36 FTL 22745.35LOC100130553 22658.54 LOC728658 22562.38 ACTG1 22481.09 LOC64460422386.22 RPL38 22292.94 LOC642210 22201 LOC148430 22112.95 LOC10013346522026.54 LOC400963 21937.41 TMSB10 21854.66 LOC284393 21771.97 RN7SL121682.27 RPS29 21599.25 LOC100133931 21519.28 RPS12 21431.97 ITIH521348.51 LOC341457 21263.76 LOC642250 21185.09 KIAA0101 21102.53LOC389435 21019.57 LOC100132488 20946.02 ANXA2 20864.81 LOC44103420786.82 RPS19 20710.05 ACTB 20640.19 LOC440589 20572.73 LOC38872020496.3 LOC728658 20419.73 FAM177A1 20346.14 LOC387930 20271.08LOC440595 20196.18 LOC653232 20127 ORC6L 20058.03 RPS25 19986.5 ACTB19922.52 LOC100130980 19848.39 PDCD7 19772.26 RPL18A 19703.81 LOC64289219628.75 LOC727808 19558.66 LOC389223 19478.59 ROCK2 19416.46 CCR619350.04 RPS15A 19290.37 RPS11 19219.96 RPL18 19149.27 RPL6 19086.13RPS29 19022.98 RPL38 18959.29 RPL27A 18894.7 RPS27 18834.63 LOC38784118770.86 TUBA1A 18706.36 LOC647361 18639.92 RPL11 18576.44 LOC10013044618517.91 LOC728553 18455.8 UBA52 18391.57 LOC728576 18328.88LOC100129553 18269.35 MYL6 18203.91 ACTG1 18140.81 FTHL16 18076.95LOC441876 18016.04 LOC343184 17954.72 LOC391777 17893.68 RPL9 17830.68RPS27A 17768.43 LOC643863 17711.83 RPS6 17652.27 PSMD12 17594.21 MYL617535.65 LOC646195 17476.44 RPL32 17418.94 RPLP0 17356.64 LOC72940217294.36 RPS17 17236.98 RPL35A 17181.16 RPL11 17118.82 RPL18A 17060.66LOC100129158 17003.18 LOC401019 16944.5 LOC100133607 16889.64 LOC72932416834.91 LOC645895 16775.01 LOC649076 16721.19 RPS16 16662.93 PPIAL4A16607.66 RPL17 16550.82 RPS10 16491.53 LOC440733 16435.98 RPL18A 16384.7LOC645899 16331.75 LOC644029 16276.86 VIM 16220.75 LOC100129902 16168.98LOC440027 16117.11 LOC728517 16063.24 C10orf58 16009.07 LOC72836815951.03 LOC439953 15892.32 LOC651894 15837.98 LOC644745 15784.83 FARSLB15733.5 LOC388524 15675.03 FTHL7 15621.63 RPL27 15573.05 RPL12 15524.71RPL37A 15473.36 RPLP1 15423.13 RPS20 15371.34 RPS14 15315.83 XPNPEP315265.39 LOC389101 15209.69 LOC402057 15164.27 LOC643509 15114.24LOC284230 15063.66 LOC642357 15015.66 LOC652071 14963.6 RPL39 14914.66LOC728576 14864.7 LOC644464 14816.74 ACTB 14769.08 RPL30 14718.38 RPS314668.78 IFITM2 14620.77 LOC644039 14568.97 LOC645683 14518.14 FAM115A14471.98 RPL19 14423.17 LOC653162 14375.34 LOC441775 14323.48 LGALS114273.15 RPS24 14227.73 RPL3 14179.84 RPS15 14130.03 GNG11 14079.57 CAV114033.15 LOC729090 13979.81 TM4SF1 13927.96 NACA 13881.36 ATP5EP213834.41 LOC653881 13785.78 RPL17 13738.14 OAZ1 13689.32 LOC64529613641.2 S100A10 13595.26 RPL31 13545.91 RPL24 13494.67 LOC10012850513442.05 LOC645174 13394.88 LOC648729 13345.11 RPL8 13298.89 LOC73109613253.78 LOC642741 13211.35 LOC647276 13169.46 LOC729903 13121.3LOC401019 13076.43 LOC728128 13029.34 CD81 12985.19 LOC731542 12939.34LOC651436 12894.54 RPL10A 12850.49 LOC441013 12803.59 RPS4X 12755.92GJC1 12710.73 LDB2 12664.08 RPS8 12614.56 BTF3 12567.71 WBP5 12522.85LOC650276 12477.88 PFN1 12433.05 RPL6 12389.93 LOC100129141 12346.77ATP5B 12304.98 CD93 12259.89 VIM 12217.76 LOC730754 12172.71 HSPB112127.76 LOC728453 12085.1 EIF4A1 12041.45 LOC389404 11999.19 CD15111957.83 LOC647276 11912.95 LOC729789 11868.46 LOC728937 11828.98 IFITM311788.35 LILRB3 11744.43 LOC646294 11705.26 RPS2 11661.18 FSCN1 11621.16LOC441246 11578.21 LOC645387 11538.01 LOC647099 11499.43 PRCP 11460.34SOX18 11422.95 LOC440575 11382.54 LOC641814 11344.01 KCNH6 11306.56LOC653314 11266.4 LOC100133649 11225.89 LOC729603 11184.33 TUBA1C11147.33 LOC286444 11110.15 LOC643531 11068.19 LOC643284 11030.72 AP2S110993.52 PTRF 10952.09 H3F3A 10913.04 LOC100132742 10876.23 LOC64821010835.95 EIF3E 10798.77 RPL3 10762.44 TXN 10725.63 RPS29 10688.26 MYL12A10651.34 GABPB2 10615.8 RPS9 10581.37 ATP5EP2 10543.17 LOC64700010509.38 UBB 10473.09 LOC388556 10436.98 LOC728693 10403.82 NGFRAP110370.85 COX7C 10337.94 GAPDH 10305.26 GNAS 10271.86 LOC400721 10233.35ICAM2 10200.27 RPS3A 10164.91 LOC100131713 10128.06 B2M 10097.39 UBB10066.7 CLEC2D 10033.68 MGC26356 10004.77 LOC100133273 9971.113 TPT19939.753 RPSA 9911.836 RPS13 9882.49 ENO1 9852.533 LOC100132742 9822.571LOC729617 9790.951 S100A10 9759.989 LOC730187 9729.674 LOC6480009697.648 LOC644464 9669.355 RPS5 9640.032 RPL14L 9608.673 RPL36AL9578.568 NEDD8 9548.513 RPS6 9520.098 TGFBR2 9492.364 RPLP1 9463.913LOC440926 9432.986 TUBA1B 9407.654 LOC284821 9377.909 BTF3 9350.254LOC730246 9325.183 LOC731365 9295.564 LOC729466 9265.775 LOC6462009235.456 ADAM15 9210.81 RPS27L 9182.401 AKR1D1 9154.34 CYB5R3 9128.27RPS3A 9101.282 RPS4X 9076.407 CREB1 9049.403 PDE4C 9022.809 TFPI8997.954 LOC728782 8971.264 LOC645387 8944.967 SEPT2 8917.304 GLTSCR28894.782 SLC25A5 8867.191 LOC646294 8843.528 PECAM1 8815.434 H3F3A8791.886 LOC649548 8767.153 POTEF 8740.947 TGFBR2 8714.794 VWF 8689.385ITGB1 8666.613 LOC729301 8641.795 LOC100133812 8618.085 EIF3L 8594.98LOC642947 8573.153 DNCL1 8550.102 TFPI 8526.81 CDKN2AIPNL 8504.479 VAMP58479.908 CDH5 8455.61 LRAP 8433.076 RHOC 8409.996 CDKN1A 8386.336 S100A68366.138 LOC645385 8343.072 SNRPD2 8321.468 ATP5A1 8298.988 LDHA8275.572 EEF2 8253.723 LOC389141 8231.352 COX4I1 8212.02 RPS9 8190.523LOC650152 8168.617 CDC37 8145.008 LOC643358 8121.837 LOC1001332338102.312 YWHAQ 8082.7 SNX3 8061.547 AV762104 8040.466 H3F3A 8017.827PFN1 7996.922 GAPDH 7973.344 YWHAZ 7953.511 RPS14 7934.452 RPL157915.846 FAU 7885.26 GPX1 7885.26 LOC728620 7854.277 RPL12 7833.73LOC648294 7816.09 SLC16A12 7795.425 LOC645715 7775.663 RPS3A 7756.622ALDOA 7737.847 CDK2AP1 7718.065 TCEAL4 7699.396 EEF1G 7679.361 LOC6467667659.651 LOC100190938 7640.343 C21orf55 7621.212 EIF4G2 7602.998LOC100128731 7583.863 LOC100133177 7566.641 ZNF430 7547.497 CCNI7529.735 RPL36 7510.359 SERPINB6 7494.061 LOC285053 7475.076 EEF1B27455.902 C11orf10 7436.825 CALM3 7420.417 LOC441087 7403.128 TUBA1A7386.972 ZMAT3 7370.062 KLF6 7351.817 LOC643031 7336.027 PABPC1 7319.402FKTN 7301.646 CFL1 7282.935 LOC644863 7265.614 RPS27A 7248.343 RPS177229.865 COX7A2 7213.416 RPS15A 7198.388 LOC100134134 7180.453 ATP5G27162.671 IL18 7144.369 LOC283412 7127.359 NUCB1 7110.135 LOC7297987092.872 LOC387867 7076.424 PCBP1 7060.549 MRLC2 7043.793 LOC3895177027.69 LOC399900 7013.314 SERF2 6997.182 EEF1B2 6981.873 MARCKS6967.221 NACA 6951.604 LOC100129424 6936.077 NPC2 6920.058 RPL35 6904.67GPX4 6889.884 C10orf58 6874.525 TOMM7 6860.304 SLC25A3 6845.603 MDH26832.719 RPL26 6817.137 ATP5I 6800.876 CTNNA1 6785.87 RALA 6771.026FAM69B 6754.661 CALM1 6738.351 SHCBP1 6723.184 SRP14 6708.369 LOC6465316693.502 TACC1 6679.065 DPYSL2 6663.472 LOC100127993 6648.476LOC100130168 6634.481 LOC285900 6620.755 RPL7L1 6604.924 PCBP2 6590.635PNPT1 6575.888 HCG2P7 6562.18 GPR116 6548.164 H2AFZ 6534.282 COX6C6521.256 ANXA5 6508.408 NQO1 6495.337 DAD1 6480.547 COL4A1 6468.127ATP5A1 6454.348 ZNF549 6440.91 MYH9 6426.936 SEC61G 6412.935 FKBP1A6399.774 ARPC2 6387.262 EIF4A2 6373.784 EMP1 6360.16 LOC729102 6346.829DDX5 6333.568 HINT1 6318.728 LOC645436 6305.671 NOP10 6292.858 PMP226279.687 PSMB1 6265.957 SQSTM1 6253.051 LOC653737 6240.21 HSPA8 6226.94TUBB 6214.39 SHANK3 6201.642 UQCRH 6188.65 LOC730313 6176.263 ATP5L6163.733 LOC100133372 6150.941 LOC550643 6139.155 TXN 6126.585 DBI6113.103 TMBIM6 6100.574 SEPT2 6088.34 LOC642489 6075.764 CDAN1 6062.012PLSCR3 6050.002 LOC649049 6038.353 JUND 6026.033 YWHAH 6013.875 GSTP16002.531 HSP90AA1 5991.17 SLC44A4 5979.221 PSAP 5967.178 CLDN5 5954.744LOC100130445 5943.334 HNRNPD 5931.25 SOD1 5919.922 EEF1AL7 5909.133LOC647856 5897.997 TM4SF18 5886.761 PTBP1 5875.687 RAN 5864.145 RPL45852.968 RAC1 5840.897 CSTB 5829.299 C14orf156 5817.476 NME1-NME25807.022 ITM2B 5796.124 BGN 5785.701 SCD 5776.214 LOC645317 5764.427CMTM7 5753.074 TOMM7 5741.794 SEC61G 5730.923 PPM1F 5719.127 SLC25A35709.5 ACVRL1 5699.607 COMMD6 5689.162 CLIC1 5677.854 C17orf45 5667.08PRDX1 5656.452 SAT1 5645.519 SEPT9 5634.972 ATP6AP2 5624.128 CSDA5613.424 PRDX1 5602.954 BTG1 5592.841 MTCH1 5582.283 LOC134997 5573.499LOC286444 5563.255 RPS18 5552.792 HLA-E 5543.25 EDF1 5532.461 ITGB15522.71 MGST2 5511.287 EIF3L 5500.726 TM4SF18 5490.636 NONO 5480.652ECSCR 5470.367 PSAP 5460.726 PSMA6 5451.676 MARCKSL1 5442.01 LOC7297425432.988 LOC100131387 5423.154 NGFRAP1 5413.279 MAP4K2 5402.065 BEXL15392.217 TBCA 5382.14 EIF1 5371.946 MCART1 5360.794 MCM8 5351.053 PSMB65340.972 DAZAP2 5331.065 QARS 5320.504 LOC440055 5310.703 APLNR 5301.342RPL13A 5290.971 C14orf85 5281.173 CNN3 5271.19 LOC100132795 5260.873LAMA5 5251.119 SLC44A1 5241.342 LOC100131609 5232.272 ARL16 5222.225LOC100129362 5212.558 WSB1 5203.553 TSPO 5193.677 LOC645173 5184.411PRCP 5175.037 ESD 5165.838 HNRNPD 5156.636 LOC648771 5148.032 CST35139.557 PRKAR1A 5130.244 EPAS1 5121.617 HSPA8 5112.767 TPI1 5103.14NFIB 5094.79 LOC646942 5086.126 NCOA4 5077.186 FLOT2 5069.23 LOC7299785060.045 IGFBP4 5051.913 LOC650646 5043.719 ANP32B 5034.827 CCND15026.154 ATP5J 5017.307 SHFM1 5008.422 ATP5O 5000.003 LOC440927 4992.161RHOA 4983.717 H2AFY 4975.798 CTGF 4966.956 LOC728809 4957.689 RALB4949.349 FABP5L2 4940.152 NDUFB2 4932.081 LOC646483 4923.669 GNAI24916.183 MRPL33 4908.738 DDB1 4900.69 LOC441073 4892.921 LOC6494474885.438 WDR1 4877.46 LOC400948 4868.795 TIMP1 4860.48 LOC4022514852.983 GNB1 4845.2 RPL36AL 4837.16 NOP10 4828.969 CHCHD2 4821.331HIST1H4C 4813.682 FLJ46309 4805.906 ANGPT2 4798.277 C20orf52 4790.903HOXB5 4782.823 NDUFS5 4774.766 UQCRQ 4766.396 LOC402694 4758.111LOC644914 4750.406 CXCR4 4742.489 WBP2 4733.885 COX5B 4725.663 LOC6466304717.094 PRDX5 4704.898 RPS26P11 4704.898 NFKBIA 4694.165 LOC7284814686.756 LOC100128084 4679.009 SRGN 4670.712 LOC645452 4663.261 ARHGDIB4655.951 SUMO2 4648.817 TRAM1 4641.106 LDHA 4633.259 MYH9 4625.853CTGLF7 4618.362 LOC388654 4611.233 CALM2 4604.143 POFUT1 4596.614 HDAC14588.697 ROMO1 4581.606 SHANK3 4574.51 RPL5 4566.714 NDUFAF3 4559.808GSTO1 4551.681 SRP9 4544.382 CCDC72 4537.007 EIF3F 4529.952 SRGN 4518.68RPS6P1 4518.68 PFDN5 4508.432 SCARB2 4500.946 ESAM 4494.812 HNRNPAB4487.656 EGLN2 4480.514 LOC401537 4473.142 EMP3 4466.904 COX5A 4459.584SHC1 4452.944 LOC648249 4445.896 ANXA1 4438.55 LOC728428 4431.064 COMMD74424.43 LOC392437 4417.459 CSNK1E 4410.571 GNS 4403.982 LAMP1 4397.37DNAJA1 4390.667 SPARC 4384.334 SNRPG 4377.093 RNF7 4367.271 LOC7296794367.271 CAP1 4356.992 LOC613037 4350.417 FAM129B 4343.163 PRDX54336.744 SNHG5 4330.19 LILRB1 4323.835 ATP5J 4317.569 CCT7 4311.306VDAC3 4304.92 GIMAP8 4298.598 PTTG1IP 4291.833 PEA15 4285.228 MDK4279.023 LOC728672 4272.423 LTA4H 4265.782 ARPC3 4259.694 SFRS5 4253.734FABP5 4247.866 B2M 4241.581 JAM3 4235.323 ATP5H 4229.135 ZFAND5 4223.899UBE2E1 4217.975 LOC100128266 4211.829 NDUFA1 4206.047 FKSG30 4199.931TUBB 4194.265 LOC389168 4188.835 C21orf24 4182.646 PAM 4176.248LOC647340 4170.147 ZNF14 4164.111 MIF 4158.17 COX6B1 4151.975 NDUFS84145.751 SF3B14 4139.69 LOC389168 4132.783 PSMD10 4126.234 ATP5H4119.575 LOC644315 4113.929 LOC643357 4107.532 COX8A 4101.995 HSP90B14095.739 SAE1 4090.083 YWHAB 4084.048 LOC390345 4077.921 RPS26L 4072.481SFRS6 4066.232 CMTM3 4060.137 NDUFB8 4054.134 RPL7A 4048.417 LASP14042.834 LOC730029 4037.351 GSTO1 4031.487 HMGN1 4026.092 HBXIP 4020.532LOC390557 4014.99 KLF6 4009.341 RTN4 4003.429 AP2S1 3997.423 TMEM173991.647 LOC100132795 3986.28 DYNLL1 3980.465 UBE2D3 3973.772 LOC927553968.05 GPX4 3962.772 APLN 3956.977 LYVE1 3951.747 AHNAK 3946.472LOC652624 3940.562 ATP6V0E1 3934.799 EIF4G2 3929.242 FAM43A 3923.254LOC728873 3917.709 PFDN5 3912.337 LOC440737 3906.923 HNRPM 3898.701CYB5B 3898.701 LOC728126 3890.699 RALGDS 3885.374 GIMAP4 3880.145 PPP2CA3874.804 CIRBP 3869.011 C9orf80 3863.877 CD34 3858.429 ATP6AP1 3852.803MRFAP1 3847.485 LOC649821 3842.339 EZR 3837.019 C20orf24 3831.573 CD99L23825.978 AIRE 3820.74 PSMC1 3815.655 C10orf10 3810.431 LOC23117 3805.025ATP1A1 3799.669 TKT 3794.411 PSMB7 3789.396 JUP 3784.532 LOC6434333779.673 TMEM66 3774.548 PSMC1 3769.582 NDUFA3 3764.494 MAGED1 3759.579C20orf24 3754.453 LOC646785 3746.863 LOC653226 3746.863 SET 3739.565CRIP2 3734.717 GLRX5 3730.108 LOC100131196 3725.29 PGD 3720.447 TCEB23715.75 BX537698 3711.047 TMEM59 3706.181 C8orf37 3701.639 ZNF4283696.942 PHLDA1 3692.216 TUBA1C 3687.356 ERP29 3682.382 RPL21 3677.297ESM1 3672.187 LOC728139 3666.905 FAM50A 3661.865 LAMC1 3657.38 UBE2I3652.873 ACTR2 3648.318 RPS15A 3643.939 C8orf45 3639.256 B4GALT5 3635.08ADD1 3628.227 FAM119A 3628.227 LOC646819 3621.287 EIF3B 3617.007 C6orf483611.933 HLA-A 3607.342 ALKBH5 3602.68 KHDRBS1 3598.262 LOC1001346483593.131 SNRK 3588.844 MAPRE1 3584.246 APP 3579.695 ATP5F1 3574.877DYNLRB1 3570.567 RASIP1 3565.849 LOC729926 3561.556 CS 3556.809 NUCKS13552.277 C20orf100 3547.784 SFRS5 3543.466 FCGRT 3539.09 ALDH9A13534.536 JTB 3530.451 DCTN2 3525.898 FAM127A 3521.184 EPN1 3516.76LOC402112 3512.346 RRAGA 3507.74 ARHGEF2 3503.294 ITGB1 3498.718 FKBP1A3493.963 NDUFA12 3489.713 VEGFB 3485.396 FEZ2 3480.915 FKBP1A 3476.397TRMT112 3471.984 PRKCH 3467.297 LOC391370 3462.874 RAB11A 3458.473S100A16 3454.14 ROBLD3 3449.96 TALDO1 3445.991 RPL22 3441.584 LOC6445113437.516 LOC127295 3431.188 ANXA2 3431.188 ARF4 3424.504 AHR 3420.322TXNDC5 3415.883 LOC646688 3411.785 NDUFB3 3407.559 AP1S2 3403.573 DNAJC83399.217 SFRS2 3394.83 MATR3 3390.652 ATP6V0C 3386.519 C3orf34 3382.759NDUFB3 3378.886 LOC728553 3374.77 HNRPA2B1 3370.461 OCIAD1 3366.672TMEM14C 3362.56 IGFBP2 3358.728 VAMP8 3354.294 UQCRFS1 3350.384 EIF3D3346.006 CTNNA1 3341.683 SQLE 3337.535 TSPAN18 3333.603 RNASE1 3329.575CD99 3325.701 ATP6V1E1 3321.748 OSTC 3318.133 PRR13 3313.96 HNRPA1P43310.016 LOC440353 3306.287 ERGIC3 3301.974 C2orf69 3297.976LOC100133477 3293.898 LOC728698 3289.942 LOC648390 3286.072 HK1 3282.036PDHB 3278.294 FLJ44124 3274.603 TUG1 3270.77 MORF4L2 3266.977 AL157483263.189 MYADM 3259.222 DEGS1 3255.744 LOC727865 3251.966 LOC7292363248.111 LOC158345 3244.281 PARK7 3240.659 CS 3236.723 BMS1P5 3233.004LOC390354 3229.485 SNRPB2 3226.027 PCBP2 3222.358 LOC440043 3218.805LOC402175 3215.17 TMBIM4 3211.304 LOC730004 3207.415 LOC374395 3203.745ZDHHC8 3200.006 MFNG 3196.474 AMY1C 3192.522 VCL 3188.645 GABARAPL23185.102 TUBB2B 3181.562 RCN1 3176.229 PTK2 3176.229 C14orf173 3170.849LOC399804 3166.942 VKORC1 3163.391 CCNY 3159.724 PRNP 3156.361 PTP4A23152.617 NDUFB5 3148.956 LOC100131801 3145.122 NDUFA4 3141.587 GAPDH3137.906 MRPS21 3134.596 HSPD1 3131.109 DARS 3127.618 PLOD1 3124.315LOC347544 3120.537 DUXAP3 3116.969 POMP 3113.4 GPIHBP1 3109.723 PLS33106.332 PGK1 3101.494 ITPR3 3101.494 HIATL2 3096.506 ZNF486 3092.927MFSD10 3089.188 PON2 3085.436 NME1 3082.096 LOC731985 3078.831 ILF23075.404 DSTN 3071.802 SFRS9 3068.507 DUSP19 3064.885 GHITM 3061.454FAM124B 3057.985 ATP6V1E1 3054.703 PTTG1IP 3051.21 HPCAL1 3047.698ZNF394 3042.981 RAB7A 3042.981 CAV1 3037.999 DYNC1LI2 3033.362 FASN3033.362 PTBP1 3028.638 CCDC130 3025.44 PSMA4 3022.079 HMGN1 3018.673TMED3 3015.24 CCT8 3011.764 IL10 3008.282 LOC645058 3005.028 MORF4L13001.861 SLC44A2 2998.583 TMEM123 2995.415 MAT2A 2992.155 ADM 2988.951PRND 2985.625 HNRNPK 2982.658 NOL7 2979.434 YBX1 2976.279 LOC3916562973.021 CAMLG 2969.814 FLNB 2966.365 ARL6IP1 2963.044 LOC3999882959.773 LOC100130562 2956.718 RWDD1 2953.291 LOC650518 2949.922 TCEAL32947.075 S100A4 2944.066 EIF2S3 2940.487 PRDX6 2937.178 TSPAN9 2934.115GLO1 2931.327 PSMD6 2928.123 ILK 2924.745 ACADVL 2921.625 RHOC 2918.659PSME1 2915.555 LOC387820 2912.308 LDLR 2909.147 TPM2 2904.888 LOC7288882904.888 SEC11A 2900.347 TEAD2 2897.152 SLC25A6 2894.058 BTBD2 2890.722NCL 2887.878 LOC100132391 2884.602 RPN1 2881.471 TRIM8 2878.486 HEXB2875.395 ZMAT3 2872.252 MGST3 2869.117 APP 2866.02 LOC728244 2863.085ARGLU1 2860.002 LEPROT 2857.123 DDX51 2854.073 CXXC5 2850.969 AP1S22847.996 LOC653314 2845.126 SRP14P1 2842.19 ACP1 2838.992 C14orf1532836.086 C20orf30 2832.782 UBA1 2829.927 SNRPB 2826.874 TXNIP 2823.728NUDT14 2820.921 LOC642817 2818.102 ATP1B1 2815.268 CSNK2B 2812.341 SNRPF2809.605 UXT 2806.481 EIF3M 2803.284 ALDOA 2800.582 EFEMP1 2797.68 STAU12794.503 ANAPC13 2791.876 DMC1 2788.981 HNRNPH1 2785.906 LPP 2783.35KRTCAP2 2780.536 RPL14L 2777.793 RPRC1 2774.822 DKK3 2771.896 BUB32769.109 CAPZA2 2766.271 MGC16121 2763.458 EIF4B 2760.512 MYH10 2757.669LOC100134159 2754.867 ARL2 2751.979 COLEC12 2749.382 RHOJ 2746.622LOC401115 2743.952 TIMM23 2741.358 CARM1 2738.743 PJA2 2736.055 CMIP2733.33 TINP1 2730.681 COPA 2728.078 SSR4 2725.157 LOC645688 2722.435PALM 2719.394 UBE2D3 2716.675 TMSL3 2714.115 EID2B 2711.423 TGM22708.749 P4HB 2706.261 NAT5 2703.492 LOC653079 2700.971 STX16 2698.241PUF60 2695.483 SEC61B 2692.776 KLF2 2690.108 LOC441506 2687.565 PSMA12684.755 DAPP1 2682.076 RAB10 2679.581 TIMP2 2677.038 NDUFA8 2674.402PRDX5 2671.801 PSMA5 2668.989 PIGY 2666.487 PRSS23 2663.739 ATP6V1F2661.253 C2orf28 2658.504 PLS3 2655.947 STARD7 2653.165 FDFT1 2649.481LOC100130003 2649.481 NUP62 2645.797 PSMB3 2643.409 FAM39E 2640.664LOC653505 2637.95 TOMM6 2635.176 AK095855 2631.407 CAPNS1 2631.407LOC649553 2627.467 RPL17 2625.058 RBX1 2622.313 CYBA 2619.898 ARPC1A2617.495 VAMP3 2614.954 LOC100133772 2612.487 SUMO3 2609.9 CD34 2607.309PRMT1 2605.064 CD63 2602.476 TPI1 2599.884 BRI3 2597.245 LMNA 2594.722SNRNP70 2592.098 ID3 2589.513 LOC442454 2587.179 CAV2 2584.557 POLR2G2582.077 LOC388707 2579.665 ATP6V0E1 2577.161 LOC654194 2574.714 PHPT12572.017 POLR2F 2569.517 APEX1 2567.108 EIF3K 2564.684 LOC6532262562.343 C15orf24 2559.838 IMPDH2 2557.397 DUSP3 2553.815 KPNB1 2553.815NDUFA11 2549.928 CTGF 2547.565 NDUFS4 2544.957 BLZF1 2542.604 RHEB2540.184 PRICKLE4 2537.998 PTPLAD1 2535.534 HSP90AA1 2533.024 BANF12530.587 COL4A5 2528.138 SDCBP 2525.598 LRRC37B2 2523.156 LRRC322520.757 GSTM1 2518.524 TTC3 2516.152 DYSF 2513.921 ETS1 2511.662 PON22509.463 PDCD6 2507.209 TOMM20 2504.969 REPIN1 2502.723 BOLA2 2499.166LOC391126 2499.166 SIVA1 2495.534 HPRT1 2493.245 PRDX3 2491.116 CDC162488.933 ATOX1 2486.616 RBM22 2484.209 NUAK1 2481.962 VPS29 2479.728VDAC1 2477.395 EVL 2475.158 TAGLN2 2473.013 LY6E 2470.802 GPR56 2468.456REEP5 2466.211 ZNF69 2464.034 LOC728590 2461.808 EEF1D 2459.394 POLR2H2457.249 PPP2R1A 2454.819 PSMB5 2452.568 C20orf43 2450.369 SPCS12448.261 ATF4 2444.895 EIF4A1 2444.895 OSTC 2441.645 RASGRP3 2438.988LOC100128353 2436.823 LOC648024 2434.574 EHD4 2432.192 VPS26A 2429.896ARAP3 2427.613 SDHB 2425.444 RPS6KA2 2423.269 MRPS6 2420.95 LOC6482102418.767 EIF3G 2416.711 CNBP 2414.27 GPR56 2412.085 SH2B3 2409.804 REXO22407.484 RNF181 2405.349 TSPAN3 2403.161 ADCY4 2401.073 HNRPUL1 2398.99LOC388339 2397.042 DDX3X 2394.857 GALK1 2392.643 PPP1CC 2390.58 HSP90AB12388.513 FAM107B 2386.169 CREB1 2384.065 VIL2 2381.946 SNRPF 2379.903TST 2377.882 LOC730534 2375.733 MKNK2 2373.554 STC1 2371.352 EIF3I2369.413 PPP1R11 2367.293 MYLIP 2365.426 SNRPB 2363.423 SDCBP 2361.481PSMB4 2359.383 YY1 2357.516 NDUFS3 2355.493 H1F0 2353.539 THBS1 2351.476SMS 2349.239 LOC391075 2347 ARF1 2345.002 ZMIZ1 2343.074 RHOG 2341.097EIF4H 2339.187 RAC2 2336.985 PPA2 2334.924 MSN 2332.827 RPL23 2330.874ITGB4BP 2328.691 MYL6B 2326.759 MFGE8 2324.612 ADSL 2322.313 RPL10A2320.322 SHISA5 2318.275 SGSM2 2316.145 ARL5A 2314.145 LOC6440632312.122 DHX15 2310.085 LOC642956 2307.807 DDOST 2305.894 SDHALP12303.914 EIF4H 2302.04 MRPL22 2300.216 LOC100132528 2298.284 LOC6536582296.237 DYNC1I2 2294.276 C20orf30 2292.387 HNRNPR 2290.423 G3BP22288.484 ZNF682 2286.593 PGRMC1 2284.582 LOC728492 2282.552 BAX 2280.554MGC4677 2278.547 NNAT 2276.699 SRRM2 2274.895 GUK1 2272.81 S100A42270.843 TMEM14C 2268.872 TIE1 2267.041 IL32 2265.217 RPS27 2263.252GSTM2 2261.182 SUMF2 2259.346 DDT 2257.489 C20orf199 2255.455 ARCN12253.645 CSNK1G2 2251.65 UCHL1 2249.632 MDH1 2247.564 ARL2BP 2245.594TEK 2243.715 TCEB1 2241.851 M6PRBP1 2239.955 CAV2 2238.078 HYAL22236.148 PRKCDBP 2234.444 NUDC 2232.664 NBPF10 2230.705 NDUFA2 2228.844LXN 2227.013 LOC647000 2225.325 C20orf160 2223.387 PPM1G 2220.516 UBL52220.516 URM1 2217.626 VASH1 2215.82 XRCC6 2213.876 PSMB2 2212.134TCEAL8 2210.404 ZNHIT1 2208.435 SETD3 2206.564 NHP2 2204.765LOC100128288 2202.999 SNRK 2200.977 SDPR 2199.231 ESYT1 2197.376 GDI22195.532 LOC100130561 2193.647 CUTA 2191.759 GJA4 2189.946 SFRS42187.892 TMED9 2186.194 CATSPER2 2184.456 RAI14 2182.639 PSMD10 2180.94AB074172 2179.276 UQCRHL 2177.506 ARMET 2175.753 MAP1LC3A 2173.799ZNF652 2172.067 TPM1 2170.117 LMO2 2168.384 WDR18 2166.626 PODXL 2164.78PCMT1 2163.02 FERMT2 2161.297 SNHG6 2158.576 ACLY 2158.576 TMEM982155.966 GYPC 2154.276 HNRNPM 2152.428 FAM171A1 2150.789 PXDN 2148.125NGRN 2148.125 LSM2 2145.52 CIB1 2143.783 NDUFB7 2142.01 ANKS1A 2140.256NPTN 2138.669 EFNB2 2137.024 C12orf57 2135.199 PRDX4 2133.33 BEX42131.534 RDX 2129.679 TPM1 2128.18 LOC391811 2126.614 HNRNPA0 2124.898RAB5B 2123.318 AARS 2121.396 RBM10 2119.742 CKLF 2117.921 C15orf632116.23 ARPC5 2114.352 DAB2 2112.622 HLX 2110.92 CD46 2109.263 ARHGAP232107.661 ERH 2105.924 GLTP 2104.153 OXA1L 2102.456 ADAMTS9 2100.784TUBB6 2098.927 PRPSAP1 2097.122 LOC728533 2095.487 CETN2 2092.925 COMT2092.925 MIR1978 2090.444 ATP1B3 2088.676 TCF25 2086.963 GSPT1 2085.414LOC728031 2083.839 HOXB7 2082.164 LOC644907 2080.516 XPNPEP1 2078.884ZNF22 2077.23 DPY30 2075.5 LOC653773 2073.876 LOC100128410 2072.218CCL14 2070.749 LOC648622 2069.157 CRCP 2067.613 COMMD3 2066.041LOC728661 2064.404 FEZ2 2062.769 QRFPR 2061.197 CD2BP2 2059.596LOC645138 2057.918 ANXA2P1 2056.255 ACTN1 2054.614 SASH1 2052.96 TPM22051.325 RBM5 2049.709 CTSL1 2048.076 HSPD1 2046.443 LOC728554 2044.892TEK 2043.162 LOC148430 2041.445 CLTA 2039.745 DDX1 2038.117 MGC878952036.414 ENY2 2034.704 C21orf58 2033.058 EIF3H 2031.374 PTOV1 2029.659C19orf10 2027.986 NSA2 2026.356 SLC2A3 2024.764 PRDX3 2023.325 GTF2A22021.712 BCKDK 2019.916 SPTLC1 2018.472 SPCS2 2016.07 C17orf61 2016.07C13orf15 2013.725 GRAP 2012.201 TXNDC17 2010.558 GLG1 2009.077 RING12007.525 CDC16 2005.976 FTHL12 2004.417 JMJD8 2002.931 DYNLRB1 2001.493LOC730740 2000 GTPBP6 1998.578 ADAM19 1997.024 OCIAD1 1995.423 ALPP1993.895 LOC645452 1992.252 LOC730455 1990.712 CIP29 1989.102 HDHD1A1987.7 PRDX2 1986.069 SSU72 1984.546 TBCB 1983.009 UBXN4 1981.49 LAMP21979.835 SOX7 1978.374 TSPAN4 1977.045 SH3BGRL 1975.596 JAG2 1974.152LDHB 1972.673 ANKRD30B 1971.064 STOML2 1969.434 MT2A 1967.846 CKAP41966.368 PABPC4 1965.115 COX7A2L 1963.582 BCAP31 1962.06 VPS35 1960.581LOC730316 1959.032 HSPE1 1957.463 DECR1 1956.08 NBPF20 1954.56 HDAC21953.03 DYNLT1 1951.625 MTPN 1950.182 ATP5E 1948.728 CLCN7 1947.253KDELR1 1944.949 GARS 1944.949 FNTA 1941.981 NME4 1941.981 ADAR 1939.685SS18L2 1938.165 SNHG7 1936.806 CLIC4 1935.304 MRPL22 1933.809 PLIN21932.481 NDEL1 1930.949 LOC100131531 1929.469 BC036485 1928.086 UNC84B1926.666 SEC61A1 1925.247 PPP2R2A 1923.752 CCT2 1922.433 HGS 1921.051RNASEK 1919.6 EIF4A3 1918.241 TUBB2C 1916.747 APEX1 1915.376 CCND31913.884 RPAIN 1912.433 LOC729841 1911.051 UNC50 1909.61 RPP21 1908.262LZTR1 1907.012 ABCA1 1905.602 NDUFV2 1904.214 TAF15 1902.111 F2R1902.111 GSTK1 1900.015 LSM1 1898.52 GRK5 1897.066 RPS23 1895.565 RPLP01894.206 SRPX 1892.791 SRP14 1891.423 LOC642590 1890.132 MRPL51 1888.738MTSS1 1887.426 SPG7 1886.074 ETFA 1884.622 NCSTN 1883.33 ARS2 1882.034LOC90586 1880.692 TPD52L2 1879.422 RHBDF1 1878.106 MAPK3 1876.805RSL24D1 1875.555 PIN1 1874.261 CTSB 1872.918 CCDC90B 1871.493 NAP1L41870.159 ATP9A 1868.852 EVI1 1866.765 POLR1D 1866.765 SSTR1 1864.751PCID2 1863.457 HIGD1A 1861.98 MGC10997 1860.583 STRAP 1859.285 MRPL361857.963 ARHGEF7 1856.522 ATP5D 1855.212 NR2F2 1853.965 FNBP1 1852.702LMBRD1 1851.216 LOC392437 1849.863 DNAJB11 1847.944 ELTD1 1847.944ZNF358 1846.034 PPP6C 1844.683 LOC646567 1843.41 GOLGA7 1842.093 ID11840.693 UBXN1 1839.336 IPO11 1838.036 TNFRSF10B 1836.729 C7orf591835.465 SYT11 1834.199 OSTF1 1832.859 ZNHIT3 1831.481 GOT2 1828.835LOC399748 1827.599 HSBP1 1826.317 EFNA1 1825.115 IDH1 1823.88 HNRPK1822.669 ANGPTL2 1821.346 HOXB8 1820.111 TMEM85 1818.718 SIAH1 1817.475LOC285741 1816.02 CFDP1 1814.757 LOC652489 1813.434 BANP 1812.163C2orf25 1810.833 ARHGAP17 1809.513 SMS 1808.21 ATP6V1G1 1806.998 TMED21805.879 CTDSP2 1804.656 PPP2CB 1803.45 PSMD4 1802.198 FKBP14 1800.996LUZP1 1799.739 CTSL1 1798.517 GLCE 1797.266 DCTPP1 1795.899 PCNP1794.674 MRPL37 1793.311 SSBP1 1792.031 BZW2 1790.698 GLB1 1789.417 STOM1788.173 ZYX 1786.884 EIF5A 1785.669 NUMB 1784.498 PSMD7 1783.367 FXR11782.152 PARP4 1780.857 RARS 1779.561 RBMS1 1778.275 FAM175A 1776.427SF3B1 1776.427 LOC100128936 1774.579 LOC644191 1773.38 CHRNA5 1772.196EIF2AK1 1771.002 MGC71993 1769.83 LOC255167 1768.607 CAB39 1767.443 FGD51766.278 HNRPR 1765.174 RPS26 1763.91 TAX1BP3 1762.65 PSMC5 1761.404LOC642755 1760.256 LOC202781 1758.944 DKK3 1757.831 ZMYND11 1756.637C19orf70 1755.48 SVIL 1754.196 SELS 1753.02 NDUFB11 1751.891 CPNE31750.703 MRI1 1749.53 LOC401397 1748.318 TPRG1L 1747.141 VAT1 1746.025TNFRSF1B 1744.824 C5orf28 1743.673 NOSIP 1742.474 FER1L3 1741.29 RPS281740.056 TCEAL4 1738.878 FAHD1 1737.738 PQLC1 1736.625 ATP6V1A 1734.904TAX1BP1 1734.904 WDR6 1733.152 GPS1 1731.944 DAB2 1730.771 LOC3388701729.516 HNRNPM 1728.303 PUM1 1727.184 SLC9A1 1725.915 TRMT5 1724.831ATP1B1 1723.685 EMD 1722.585 PSMG2 1720.345 CCDC23 1720.345 C20orf241720.345 UROD 1718.176 PPP2R2B 1717.048 COPB1 1715.868 PUM1 1714.76CASP2 1713.646 TFG 1712.523 LOC728820 1711.473 CHCHD9 1710.374 CSE1L1709.265 LOC100131785 1708.063 FAM120A 1706.955 TMEM111 1705.855 HMGN21704.702 TNK2 1703.603 PTPRF 1702.506 C19orf56 1701.464 C1orf85 1700.401CLDND1 1699.33 SF3B5 1698.141 CCDC56 1696.976 HIGD2A 1695.904 SRP541694.673 MRPS18C 1693.505 SLC38A2 1692.344 SYPL1 1691.19 PWP1 1690.03CYC1 1688.812 VPS28 1687.69 BSG 1686.67 TRIOBP 1685.549 IDH3B 1684.483FAM65A 1683.363 IMP3 1682.299 SCRN1 1681.224 PLOD3 1680.136 TSC22D11679.005 UCKL1 1677.916 C3orf54 1676.807 TCEAL3 1675.69 ZRANB2 1674.65SFRS18 1673.475 PSMC2 1672.436 TMEM147 1671.356 CNDP2 1670.377 BAT11669.32 PSMD4 1667.617 SYPL1 1667.617 FAM96A 1666.04 PFDN1 1664.983MRPS12 1663.866 MXD4 1662.821 PSMA6 1661.627 LOC729279 1660.481 LRP101659.336 MRPL17 1658.299 WSB1 1657.178 ARL6IP5 1656.15 HSD17B7 1655.049CYP1A1 1654.03 RGL1 1653.014 ARHGDIA 1651.931 LSM3 1650.846 LOC4403591649.8 LOC730744 1648.793 SFRS14 1647.759 LOC653566 1646.763 LOC6518941645.664 CLDND1 1644.57 PTDSS1 1643.545 SSB 1642.473 GRIPAP1 1641.337RRAS 1640.329 PRPF8 1639.153 CRTAP 1638.192 AV737317 1637.094 PDIA51635.993 GYPC 1634.996 LOC653086 1633.937 SULT1A1 1632.887 EXOSC101631.823 SEC14L1 1630.73 CMTM7 1629.708 CDK4 1628.643 SLC12A2 1627.639LOC100128062 1626.109 DUSP22 1626.109 LOC100129379 1624.099 TMEM1581624.099 SH3GLB1 1622.482 TGFBR3 1621.398 SFRS1 1620.42 C1QBP 1619.375MMS19L 1618.318 FABP4 1617.363 SYF2 1615.805 LOC647285 1615.805 POM121C1614.33 CTXN1 1613.323 PMP22 1612.342 DCTN3 1611.336 EI24 1610.259 SNURF1609.211 PFKP 1608.135 AK3 1607.133 NAP1L1 1606.141 PSMB10 1605.125 FIS11604.15 RCN2 1603.109 COPS5 1602.015 UBE1 1600.951 METAP2 1599.995 DEGS11598.954 TPP1 1597.891 TCF4 1596.551 PLD3 1596.551 SLC20A1 1595.085 BRD21593.958 GTF2E2 1592.892 CARHSP1 1591.833 KIAA1949 1590.829 PSMF11589.926 DCTN1 1588.867 LOC643668 1587.862 C11orf59 1586.41 CDC42EP41586.41 LOC729317 1584.87 ATXN2 1584.024 ZDHHC16 1582.96 KLHL3 1581.971FBXO11 1581.021 HSD17B12 1579.986 WDR54 1578.996 THOC7 1578.022LOC286157 1577.048 PGAM1 1576.049 RRBP1 1575.086 COPS3 1574.042 TGOLN21572.574 ATP5J2 1572.574 PAICS 1571.114 MYCT1 1570.123 CCNG1 1569.125EIF1B 1568.22 PTPLAD1 1567.188 GLUD1 1566.225 SRRM1 1565.277 BCL2L11564.333 SDHAF2 1563.342 TMEM126B 1562.454 COX6A1 1561.487 LOC6511981560.508 TSC22D3 1559.555 ACAT1 1558.552 LOC389787 1557.626 RALY1556.622 SSR2 1555.669 MTUS1 1554.711 RNF144 1553.795 LOC1001327271552.848 JUN 1551.928 NDUFAB1 1550.977 MCM3 1550.039 MRFAP1L1 1549.157PRNP 1548.212 COL18A1 1547.275 ECH1 1546.324 LOC650369 1545.353 CDC42EP51543.892 ZNF738 1543.892 HSPA9 1542.471 KCTD12 1541.497 SUZ12 1540.117RABGAP1 1540.117 GTF2H5 1538.645 DDA1 1537.81 BIN1 1536.906 DUSP11535.978 STK24 1535.023 ITGA5 1534.085 DDX47 1533.17 SEC31A 1532.248PNPT1 1531.378 SPTBN1 1530.478 GPSM1 1529.602 EXOC7 1528.681 PSMC61527.768 GTPBP4 1526.921 DBN1 1525.956 GLT25D1 1525.037 EIF2A 1523.676LIMCH1 1523.676 CCNDBP1 1522.32 TROVE2 1521.457 RPS26L 1520.508 AURKAIP11519.568 WBP5 1518.619 RPAIN 1517.721 TPM3 1516.876 KIAA1671 1516.034LOC653994 1515.152 DBNL 1514.297 NTAN1 1513.386 BOLA2 1512.461 TMEM441511.551 EDN1 1510.177 ITGB1BP1 1510.177 CCDC109B 1508.421 ZNF221508.421 C4orf18 1507.078 APOA1BP 1506.23 SH3BGRL3 1505.32 UBE2M1504.415 CAPNS1 1503.518 SPOP 1502.701 GNG10 1501.888 PLSCR4 1500.944TMEM181 1500.131 LOC388275 1499.324 FEZ1 1498.408 COX17 1497.494 LMNA1496.663 C21orf33 1495.672 ITM2C 1494.223 DNMT1 1494.223 ITM2C 1492.824PRR14 1492.013 CYR61 1491.16 BNIP3 1490.33 IGF2R 1489.489 SON 1488.169GFOD1 1488.169 LOC641700 1486.891 MRI1 1486.039 PROCR 1485.086 LOC7320071484.229 CTTN 1483.402 LOC644799 1482.539 RUSC1 1481.565 GRN 1480.712ITGB5 1479.823 GYG1 1478.946 MRPL23 1478.119 ASAP1 1477.166 KPNA41476.338 PSME2 1475.46 PRAGMIN 1474.691 RCC2 1473.898 INPP1 1472.989LOC100132291 1472.102 NHP2L1 1471.226 ANAPC11 1470.023 CGNL1 1470.023NFATC2IP 1468.795 IARS2 1467.883 TJP1 1467.001 LOC729768 1466.205ATP6V1B2 1465.338 LOC644774 1464.489 CSF2RA 1463.664 ANGPT2 1462.786ATP5J2 1462.035 ANAPC5 1461.197 BRMS1 1460.376 ADRM1 1459.571 C2orf281458.684 DGUOK 1457.91 SLC25A39 1456.678 CHCHD10 1456.678 DGUOK 1455.463PEBP1 1454.59 PPP1R14B 1453.763 TMEM205 1453.072 GNL2 1452.202 LOC6467231451.384 BCLAF1 1450.526 PAPSS2 1449.722 ROD1 1448.848 C8orf59 1448.044CLNS1A 1447.207 TAX1BP1 1446.445 BNIP3L 1445.532 NFKB1 1444.768LOC644934 1443.99 ADAM15 1443.15 PPP1CA 1442.281 C17orf49 1441.421CGGBP1 1440.555 AP3B1 1439.754 ARPC4 1439.019 TMEM87A 1438.202 LOC4400931437.389 PRDX2 1436.503 CENPB 1435.284 RIOK3 1435.284 PEPD 1434.051C7orf30 1433.277 SF3B4 1432.503 C7orf50 1431.741 PAICS 1430.9 DHRS71430.109 SCHIP1 1429.311 SMARCA4 1428.443 LOC391833 1427.659 CD1511426.918 C14orf112 1426.134 CCT3 1425.327 TSPAN17 1424.433 HEBP11423.707 PALMD 1422.87 PSMA3 1422.059 HCFC1R1 1420.912 FAM96B 1420.912LOC728532 1419.695 TRAPPC2L 1418.904 BASP1 1418.075 ZFYVE21 1416.821EDF1 1416.821 TMEM43 1415.619 KIAA1191 1414.827 COMMD1 1414.003 VEZF11413.213 TMCO1 1412.417 PSMD6 1411.674 PAFAH1B3 1410.928 C19orf431410.128 CWC15 1409.341 PHB2 1408.51 FAM45A 1407.765 SPTLC1 1407.045C2orf29 1406.196 PGLS 1405.486 GNPDA1 1404.659 AIDA 1403.945 FNBP1L1403.256 TCEAL8 1402.467 WFS1 1401.669 CYTSA 1400.85 IFNGR2 1400.118MRPS24 1399.314 SASH1 1398.612 LOC728590 1397.854 LSM5 1397.054 NDUFB101396.327 PTPRM 1395.602 BIN1 1394.867 MLLT11 1394.156 KLHL5 1393.377CAMK2N1 1392.62 IFI16 1391.929 RAB2B 1391.157 TSG101 1390.367 ARHGAP211389.62 TXNL2 1388.918 EIF2B4 1388.222 AKR7A2 1387.016 PPP4C 1387.016MARCH7 1385.806 EWSR1 1385.086 MGEA5 1383.891 JMJD8 1383.891 TSPAN31382.76 FAM62B 1381.996 PLVAP 1381.302 ATP1B1 1380.575 LOC2204331379.767 KIAA1751 1378.533 NOX4 1378.533 BCAT1 1377.397 TRAPPC5 1376.299DIABLO 1376.299 ATG4A 1375.226 EWSR1 1374.548 LOC100130919 1373.854 EIF61373.121 SERF1B 1372.435 C2orf25 1371.71 ISCU 1370.6 EMCN 1370.6 NAE11369.468 CBX2 1368.713 GTF3A 1367.917 FXYD5 1367.183 RNU6-1 1366.377CIAO1 1365.645 SF3A2 1364.954 LOC729217 1364.264 SMARCD1 1363.514 YPEL51362.416 MAT2B 1362.416 CD9 1361.367 CLK1 1360.635 SHE 1359.905 ARPC41359.19 ENG 1358.525 DPM1 1357.848 RPS26L 1356.814 SEC24C 1356.814 WDFY11355.688 ABCF1 1354.999 SEPN1 1354.331 CTPS2 1353.527 JAK1 1352.825IGF2BP2 1352.016 CALU 1351.325 NSUN2 1350.603 ETS2 1349.889 PSMA41349.159 NOTCH4 1348.37 PPIL3 1347.714 EBPL 1346.925 UBE2A 1346.266TMEM14D 1345.427 TSPO 1344.7 UBE2E3 1344.022 SNX17 1343.293 AHCY 1342.54APH1A 1341.847 CTTN 1341.154 SH3GLB2 1340.142 LOC646347 1340.142 TUBB31339.084 LSM4 1338.417 MCM7 1337.661 UBE2G2 1336.937 CCL15 1336.199VPS37C 1335.507 MRPL43 1334.784 AKT1 1334.044 TSC22D1 1333.376 GLRX31332.713 C3orf21 1332.065 UFC1 1331.382 DDEF2 1330.627 HNRPDL 1329.926OAT 1329.173 SAMM50 1328.509 DDX42 1327.8 TMEM189-UBE2V1 1327.136 BRD91326.459 TRABD 1325.811 LYN 1325.106 LOC100130516 1324.409 UBAP2L1323.751 LYL1 1323.043 NAT5 1322.032 C19orf43 1322.032 C22orf13 1320.994PAFAH1B1 1320.273 HECW2 1319.607 DDX39 1318.904 NSMCE4A 1318.229 NRP11317.531 NLRP8 1316.868 IFFO1 1316.188 SERPINH1 1315.531 TOMM20 1314.873SLC35B1 1314.205 BMP6 1313.502 ACTR10 1312.85 AIMP2 1312.181 PLRG11311.423 TUBB6 1310.078 SWAP70 1310.078 LOC345041 1310.078 PPA1 1308.715CUTA 1308.027 MAGT1 1307.382 LOC388621 1306.728 TSPO 1306.076 CKLF1305.451 ACTR1A 1304.807 HSPA1B 1304.1 DYNLRB1 1303.429 LOC6511491302.787 TINF2 1302.134 ACTL6A 1301.494 CNIH4 1300.809 NECAP2 1300.112AKT1 1299.402 FLOT1 1298.767 C6orf153 1298.085 CUEDC2 1297.413 AK906941296.433 LOC728903 1296.433 TXNRD1 1295.501 AFAP1L1 1294.547 CAPN111294.547 UBE2L6 1293.604 KIAA0355 1292.622 LAPTM4B 1292.622 RNU6-151291.31 TGFB1I1 1291.31 TNFRSF21 1290.303 HNRNPAB 1289.261 C14orf1661289.261 C3orf10 1288.225 MAPK3 1287.591 BUD31 1286.944 CCDC50 1286.227DPM1 1285.552 TSEN34 1284.894 FAM32A 1284.298 PTGR1 1283.698 BTBD61283.024 COQ5 1282.389 DNAJA2 1281.734 YTHDC1 1281.127 CXCR4 1280.516SNCA 1279.908 C19orf53 1279.337 TMED10P 1278.664 PIP5K2B 1278.034 CTDSPL1277.381 CSE1L 1276.724 LOC728973 1276.059 ITM2A 1275.44 SEPT15 1274.779DERA 1274.102 THOC4 1273.114 SNRPN 1273.114 ATG12 1272.123 SUPT16H1271.505 NINJ1 1270.853 TRAF3IP2 1270.277 LOC100132247 1269.647 MMP11269.005 GPN1 1268.349 C16orf61 1267.703 ZFP91 1267.065 CLTA 1266.103RBM3 1266.103 STK25 1265.098 CD99L2 1264.439 SEMA3E 1263.804 MMRN11263.233 FAM38A 1262.61 CXXC5 1261.953 FAM125A 1261.062 COPE 1261.062CNRIP1 1260.09 NDUFB6 1259.195 AKR1A1 1259.195 ATP2B4 1257.931 SF3A31257.931 ACSS2 1256.642 SGSH 1256.642 MRPL32 1255.638 FBLN1 1254.976CKAP5 1254.373 PPP1R15A 1253.721 PCDHB2 1253.122 FBXO21 1252.554TMEM183B 1251.959 TYK2 1251.349 EIF2B4 1250.723 KIAA1310 1250.124 UBE3C1249.545 ZNF207 1248.609 TOMM22 1248.609 AP2M1 1247.656 RBM9 1247.018NAGK 1246.158 SIVA 1246.158 PGAM4 1245.267 BRPF1 1244.707 LOC6532321244.138 MRPL24 1243.487 ITPRIPL2 1242.837 RANBP1 1242.19 PIR 1241.579NDUFS7 1241.024 MRPL33 1240.402 LOC731777 1239.449 ACSL3 1239.449SYNCRIP 1238.21 SCAMP1 1238.21 HSPH1 1237.276 SNORD13 1236.367 ATP5C11236.367 RNPS1 1235.512 YRDC 1234.929 FNBP4 1234.016 SLC27A3 1234.016SNTB2 1233.115 AK2 1232.525 C9orf78 1231.887 SHROOM4 1231.273 CHMP51230.644 KLHDC3 1229.734 COL5A2 1229.734 MKRN1 1228.856 CLPTM1L 1228.232FZD4 1227.591 AHCYL1 1226.997 C11orf2 1226.102 TCEA2 1226.102 SERTAD21225.225 ZNF581 1224.638 TXNRD1 1224.021 MRPS22 1223.439 COPB2 1222.825EIF2B2 1222.166 MPDZ 1221.614 RABAC1 1220.997 LRRFIP1 1220.45 CCT71219.851 LOC643336 1219.27 EIF4E2 1218.658 SNRNP70 1218.084 UNC45A1217.535 EPRS 1216.981 LOC653147 1216.359 EIF4G2 1215.815 CHIC2 1215.238RALY 1214.672 COMMD4 1214.132 HAGH 1213.555 ATIC 1212.981 SEMA6A1212.362 SDAD1 1211.776 TMEM173 1211.176 WDR61 1210.555 UQCRC1 1209.94ERGIC3 1209.292 C16orf58 1208.466 FTHL12 1208.466 TIGA1 1207.301 ITGB51207.301 ATP2B4 1206.435 HSPC268 1205.868 ACP5 1205.3 CHST7 1204.734LOC728643 1204.165 TMEM93 1203.577 RNF5P1 1203.002 IMMT 1202.47 NOP561201.878 STX5 1201.322 TXNDC5 1200.716 LOC100131905 1200.21 PLEKHM21199.673 LSM7 1199.112 SPRY1 1198.584 C19orf60 1198.021 LSM14A 1197.449SRP54 1196.849 AMZ2 1196.268 FKBP9L 1195.389 RAB8A 1195.389 SPSB31194.239 GIPC1 1194.239 SLC29A1 1193.425 MRPL3 1192.832 CNIH 1192.266FAM127B 1191.741 ATP6V0B 1191.148 ATP1B3 1190.628 IDH3B 1190.099 TFDP11189.245 TECR 1189.245 GAR1 1188.402 CDR2L 1187.799 KIAA1147 1187.189IGFBP7 1186.642 HRASLS3 1186.117 PFN2 1185.528 RPL7L1 1184.957 TDP11184.364 RASA1 1183.793 BMS1 1183.25 DRAP1 1182.717 POLE3 1182.185 NARF1181.617 EBNA1BP2 1181.081 LOC644563 1180.512 HECTD1 1179.933 ATG4A1179.092 IRAK1 1179.092 CCDC92 1178.273 SNRPA1 1177.46 CAPZB 1177.46SCAMP3 1176.606 LOC642975 1176.084 CNIH 1175.454 TRAPPC4 1174.829 NISCH1174.275 ADARB1 1173.705 ECHS1 1173.163 GSN 1172.576 GOLGA3 1171.965TMEM183A 1171.472 PREI3 1170.618 COPZ1 1170.618 RNF149 1169.794 PRKRIR1169.219 KLHL9 1168.706 RPL9 1168.134 ANKRD9 1167.583 MRPL14 1167.011CCBE1 1166.449 VBP1 1165.62 LAMB1 1165.62 C12orf10 1164.491 MRPS101164.491 TWSG1 1163.691 LOC100132585 1163.139 MED6 1162.643 GAK 1162.143HPS6 1161.618 SOX4 1161.072 CLSTN1 1160.531 TAF1C 1159.974 LIMS11159.434 TRIM44 1158.63 TNPO2 1158.63 CHMP1B 1157.806 ATP5G1 1157.298TUBG1 1156.749 NDUFV1 1156.212 MAP2K1 1155.693 NOTCH1 1155.126 UBE2F1154.596 POLR2I 1154.025 RSL1D1 1153.5 MRPL21 1152.919 LOC6486951152.101 POLR2J3 1152.101 DNAJB6 1151.286 TMEM131 1150.796 CHMP51150.222 UNC84A 1149.634 FAM84B 1149.095 SOX7 1148.576 NEDD8 1148.045CRELD2 1147.203 EEF2K 1147.203 SH2D3C 1146.37 ACSS2 1145.794 CNIH1145.304 RPL13 1144.794 ARF4 1144.238 ASAP2 1143.712 HCFC1 1143.163SLC41A3 1142.617 ARID1A 1142.087 MRPL54 1141.562 SNRPB2 1140.987 PAIP21140.448 ULK1 1139.961 CALD1 1139.447 MAPBPIP 1138.939 HARS 1138.45HCLS1 1137.98 SFRS17A 1137.448 ASH2L 1136.926 LOC441131 1136.194 AIF1L1136.194 METAP1 1135.416 TTC37 1134.883 RNASET2 1134.356 CARD10 1133.855ATP5SL 1133.309 CTSC 1132.787 GDPD5 1132.295 C5orf15 1131.76 C1orf1231131.216 MED28 1130.738 ADD3 1130.209 HES4 1129.346 VPS28 1129.346 SIRPA1128.332 PPP1R16B 1128.332 ATP1A1 1127.569 TOP2B 1127.102 CLDN141126.581 BRIX1 1126.127 GLRX 1125.649 PHRF1 1125.135 ANXA7 1124.618PEX11B 1124.07 LOC390466 1123.317 FTHL8 1123.317 RBM4 1122.305 ACO11122.305 FAF2 1121.286 ZSCAN18 1121.286 USO1 1120.278 PDCD4 1120.278BOLA3 1119.499 GIMAP6 1118.759 EXOSC1 1118.759 TNPO1 1117.953 MRPL211117.431 ETF1 1116.964 TMEM109 1116.22 PICALM 1116.22 PPP2R5E 1115.468DHX15 1115.038 RANGAP1 1114.519 NUAK1 1114.015 RAPGEF1 1113.275 C1orf431113.275 FAM38B 1112.526 ATP1B3 1111.986 AW276479 1111.51 CNPY2 1111.025CORO1B 1110.552 AV737943 1110.036 ARL6IP6 1109.565 SRF 1109.065 GALNT111108.587 DIMT1L 1108.078 SEC14L1 1107.615 PTS 1107.107 PHF5A 1106.657NIPA2 1106.208 LOC728564 1105.495 LOC728666 1105.495 CCM2 1104.829 PLDN1104.368 TMEM189 1103.863 LOC647302 1103.405 ACOT9 1102.912 EHD11102.392 TMEM88 1101.651 RIOK3 1101.651 CHMP2A 1100.961 LOC7294951100.251 LOC100129211 1100.251 C11orf74 1099.23 DHRS4 1099.23 ALDH7A11098.483 RYBP 1098.015 CISD1 1097.296 NCBP2 1097.296 PLCG1 1096.586FBXW11 1096.097 LAMP2 1095.627 C11orf67 1095.123 TXLNA 1094.675 ST131094.182 ASNSD1 1093.703 THAP11 1093.181 SCYL1 1092.677 C20orf201092.201 ANKRD11 1091.664 KIAA0494 1090.984 ATP6V1D 1090.984 MAGED11090.273 TIA1 1089.785 HPRT1 1089.321 C1orf128 1088.849 STIP1 1088.359LAPTM4B 1087.642 MED16 1087.642 SLC16A3 1086.895 EFCAB4A 1086.425 ERAL11085.952 AKR1B1 1085.496 GLIPR2 1085.028 SNRPC 1084.522 SLC41A3 1084.067C12orf35 1083.54 SMTN 1083.064 SCAP 1082.62 UBAC1 1082.171 CLINT11081.704 TNFRSF25 1081.222 MRPL45 1080.789 C4orf32 1080.313 LOC1001281961079.856 CHFR 1079.43 FUBP3 1078.945 FLJ35390 1078.417 ARSD 1077.77IMPDH1 1077.77 TSPAN6 1077.097 GLB1 1076.624 SFRS2IP 1076.142 RHOJ1075.704 TIMM22 1075.007 ARAP3 1075.007 MYC 1074.047 PRICKLE1 1074.047LOC728620 1073.366 ZNF467 1072.903 FAM160B1 1072.44 PRPF31 1071.962LOC100129742 1071.49 FOXO1 1071.02 P4HA2 1070.563 C19orf2 1070.108 XLKD11069.626 NOS3 1069.22 FXYD5 1068.745 RAB32 1068.311 CPNE1 1067.883ARPC1B 1067.446 LOC729406 1067.012 LSM3 1066.554 MYOF 1066.106 POGK1065.674 SFRS2B 1065.215 GPR137 1064.777 FAM189B 1064.313 TOMM401063.822 CRK 1063.404 DSTN 1062.951 UGP2 1062.271 ORMDL1 1062.271LOC653566 1061.591 RBMX 1061.134 ASAP1 1060.679 CDC25B 1059.996 UTP11L1059.996 U2AF2 1059.358 ARF5 1058.938 ERCC1 1058.498 VGLL4 1058.016CREB3L2 1057.327 NSL1 1057.327 AKR1A1 1056.706 NUDT5 1056.246 UBQLN11055.836 VPS41 1055.356 PDIA6 1054.718 LOC100133516 1054.718 PELO1054.057 LACTB 1053.587 XRCC2 1053.146 HIGD1A 1052.714 SEC22C 1052.285CARD8 1051.832 MAP1B 1051.378 DRG1 1050.899 STMN1 1050.489 LOC4403451050.027 DCAF7 1049.575 BOLA3 1049.18 APRT 1048.483 ZDHHC9 1048.483SFT2D1 1047.81 ZNF207 1047.343 FLJ36131 1046.928 C6orf125 1046.454 YIPF31045.998 LOC729992 1045.539 IGF2BP3 1045.127 TM9SF2 1044.687 DCTN61044.225 FXR1 1043.764 RPL34 1043.313 AP2M1 1042.912 LOC644330 1042.479TXNDC12 1042.033 BEX1 1041.572 PGM1 1041.145 NRBP2 1040.679 IRF2BP21040.249 ITFG1 1039.802 MRPL20 1039.355 MRPS17 1038.91 FAM3A 1038.477MAN2B2 1037.795 S100A13 1037.795 PTPN11 1037.141 NPEPL1 1036.503 DPAGT11036.503 STUB1 1035.88 CDK5RAP3 1035.203 LOC100128266 1035.203 LRRC411034.576 RPS21 1034.106 PIAS4 1033.669 CHP 1033.24 CPSF4 1032.79 FRMD4A1032.13 CDK10 1032.13 LOC650157 1031.44 LOC100132717 1030.745 G6PD1030.745 UROS 1030.116 BC035081 1029.698 LOC730255 1029.265 ENPP21028.837 CNN2 1028.433 OSBPL9 1027.99 TRPT1 1027.561 RN7SK 1027.133COPS7A 1026.742 NHP2 1026.284 PAPSS1 1025.863 MACF1 1025.215 ACOT71025.215 SERINC3 1024.519 LAMA4 1024.067 MRPS15 1023.652 TM9SF4 1023.053ACAT2 1023.053 LOC645166 1022.43 NCOA7 1022.016 TBC1D4 1021.59 RHOQ1021.206 FAM39DP 1020.8 TNFRSF1A 1020.394 FKBP5 1019.963 FAM120B1019.554 LCMT1 1019.166 CCDC59 1018.55 AK022936 1018.55 RPUSD4 1017.934IGFBP3 1017.538 SLC35E1 1017.154 CCDC125 1016.336 RIN2 1016.336 MBTPS11016.336 TMEM126B 1015.509 GPR177 1015.087 LOC728661 1014.67 XPO11014.219 ATG4B 1013.813 DAP3 1013.397 CISD1 1012.933 STK19 1012.524 AES1011.867 NDUFA13 1011.867 NDRG4 1011.251 FIBP 1010.835 VHL 1010.439RNF38 1009.799 PRMT1 1009.799 VPS4B 1009.162 SHMT2 1008.756 MRPL341008.353 OCIAD2 1007.943 PSMD1 1007.326 HSD17B4 1007.326 VBP1 1006.484MCRS1 1006.484 TNFAIP1 1005.872 TNRC6B 1005.446 COASY 1005.033 ST3GAL11004.617 RHBDD2 1003.993 SURF4 1003.993 KLHDC8B 1003.384 TSPAN4 1002.961KDM5B 1002.552 STK4 1002.151 LPHN2 1001.764 POLR2A 1001.35 CD59 1000.938DNAL4 1000.492 RHBDF2 1000.062

The 30-MV2-6 cells were maintained in EGM-MV2 media (PromoCell) plusTGFβ inhibitor (SB43154) (Cayman Chemical Co., Ann Arbor, Mich.) in a 5%CO₂, 5% O₂ humidified cell culture incubator. The 30-MV2-6 cells wereseeded at a density of 40 k/cm². The culture media was removed and aftertwo washes with phosphate buffered saline (PBS), (PBS) was added at 0.1ml/cm² to produce conditioned medium from which exosomes were isolated.Alternatively, basal EGM-MV2 medium (PromoCell, Heidelberg, Germany)without fetal calf serum or growth factor additives was substituted forPBS. The media was conditioned by the cells in a humidified tissueculture incubator for 16 hours at 37° C. at 5% CO₂ and 1% O₂. Theconditioned medium was collected and 0.5 volumes of Total ExosomeIsolation Reagent (Life Technologies) was added and mixed well byvortexing until there was a homogenous solution. Alternatively, asolution of 15% polyethylene glycol (Hampton Research, Aliso Viejo,Calif.), 1.5 M NaCl (Sigma, St Louis, Mo.) was substituted for the TotalExosome Isolation Reagent. The sample was incubated at 4° C. for atleast 16 hours to precipitate the exosomes, followed by centrifugationat 10,000×g for 1 hour at 4° C. The supernatant was removed and thepellet is resuspended in 0.01 volume of PBS.

Exosome particle size and concentration were measured using nanoparticletracking analysis (NTA; Nanosight, Malvern Instrument, Ltd, MalvernWorcestershire, UK) and by ELISA. The experiments were repeated usingcommercially available HT1080 cells (ATCC) as a comparison. HT1080 cellsare a human fibrosarcoma cell line known to form exosomes with vesicleforming ability (see, e.g. Kim et al. (2002) Cancer Res. 62:6312).

The results of the Nanosight NTA (triplicates) for exosome preparationsderived from 30-MV2-6 and HT1080 cells (ATCC, Manassas, Va.) are shownin FIG. 1. The results indicate that particles prepared from 30-MV2-6are from 80 to 110 nm with predominant peak at 88 nm+/−2.9 nm Theparticles prepared from a HT1080 human fibrosarcoma cells were larger bycomparison with a mode of 120 nm+/−7.4 nm The concentration of exosomesbearing the exosome marker CD63 was measured by ELISA, using samples ofknown concentrations of HT1080 exosomes as a standard curve. Sampleswere adsorbed to the ELISA plate by incubation overnight in PBS. The PBSwas removed and wells were washed 3 times in ELISA wash buffer (ThermoScientific, Waltham, Mass.) followed by incubation with primaryanti-CD63 antibody (BD Pharmingen, Franklin Lakes, N.J.) for 1 hour atroom temperature. The primary antibody was removed followed by washing 3times in wash buffer and incubation with secondary antibody (HRPconjugated anti-mouse) (Invitrogen, Grand Island, N.Y.)) at 1:3000dilution for 1 hour at room temperature. The wells were washed 3additional times with wash buffer and incubated in Super sensitive TMBELISA substrate (Sigma, St Louis, Mo.) for 0.5 hour followed by additionof ELISA stop solution (InVitrogen, Grand Island, N.Y.). Theconcentration of exosomes was determined by reading optical density in astandard plate reader at wavelength of 450 nm

Example 2 Angiogenic Activity of Exosomes Prepared from a HumanEmbryonic Progenitor Cell Line

Angiogenic activity of exosomes was assayed using an in-vitroendothelial tube forming assay. The assay was performed in triplicate ina μ well slide (Ibidi, Verona, Wis.) or in single wells of a 96-wellplate. The wells were coated with reduced growth factor Matrigel (BD,Franklin Lakes, N.J.). Human umbilical cord vascular endothelial cells(HUVEC) that were grown to 70-80% confluence were plated at 5000-7000cells per well in a μ well slide in 50 ul of EGM-MV2 basal medium(Promocell, Heidelberg, Germany) (no supplements) containing up to 10 μlof exosomes in PBS or equivalent volume of PBS without exosomes as anegative control or in 50 μl of complete EGM-MV2 medium with growthfactor supplements as a positive control. Alternatively, the assay wasperformed in a 96-well plate using 60,000 to 90,000 cells per well in280 μl of medium and 20 μl of exosomes or PBS. The cells are incubatedat 37° C. in a 5% CO2 incubator for 16-18 hours. The cells werephotographed under phase contrast at low power or stained with calceinand photographed using a fluorescence microscope. The images were scoredfor cell covered area, total tube length, number of branch points, andnumber of loops using Wimasis image analysis (Ibidi, Verona, Wis.). Atleast 3 random images were quantified per well.

FIG. 2A shows an increase in HUVEC endothelial tube formation when grownin the presence of 30-MV2-6 derived exosomes (in PBS) compared to basalmedium with an equivalent amount of PBS (with no exosomes) added(negative control). The quantified results (FIG. 2B) indicate that totaltube length, cell covered area, branch points and the number of loopswere all increased by the addition of exosomes compared to basal mediumindicating that the 30-MV2-6 exosomes are angiogenic.

Angiogenic activity of exosomes was also assessed by their ability tostimulate in vitro tube formation using human embryonic stem (hES) cellderived perivascular embryonic progenitor cells (PEPCs) (also called017-PC-A) cells bearing pericyte and stemness markers (CD146, CD133,Podoplanin)(U.S. patent application Ser. No. 14/625,621, filed on Feb.18, 2015). The assay was performed as described for HUVECs except thathES cell derived PEPCs were used instead of HUVECs. The assay wasperformed in triplicate using μ well slides (Ibidi, Verona, Wis.). ThehES pericytes that were grown in defined medium differ from HUVECs intheir response to complete medium. HUVECs respond to complete EGM-MV2medium in the tube forming assay with robust tube formation (FIG. 2A).In contrast, hES PEPCs migrate to form foci consisting of cellularaggregates (FIG. 3A) when grown on Matrigel in complete EGM-MV2 mediumand thus exhibited reduced tube formation. The hES PEPCs grown indefined medium form incomplete tubes (FIG. 3B) similar to HUVEC in basalmedium (FIG. 2A). Like HUVECs, the hES PEPCs grown in the presence of30-MV2-6 exosomes displayed an increase in tube formation as shown inFIG. 3C-3E. The tube formation was dose responsive and quantitativeanalysis indicated an increase in all 4 tube formation parameters (FIG.3F-3I). Unlike HUVECs which respond to both exosomes and complete mediumwith increased tube formation, hES derived PEPCs respond to exosomeswith increased tube formation but respond to complete medium withreduced tube formation compared to basal medium. Thus, 30-MV2-6 exosomesinduce angiogenesis in a non-angiogenic cell type that does not respondto the angiogenic factors present in EGM-MV2 complete medium. These dataindicate that 30-MV2-6 derived exosomes do not simply mimic the factorsin complete medium but instead are capable of stimulating hES derivedPEPC tube formation by a mechanism that is distinct from the action ofcomplete medium on these cells.

Example 3 Comparison of Angiogenic Activity of Exosomes Derived from aHuman Embryonic Progenitor Cell Line and Exosomes Derived from AdultBone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs)

Exosomes were prepared from an embryonic stem cell derived PureStem®cell line, 30-MV2-6, and from adult bone marrow-derived mesenchymal stemcells (BM-MSCs) from two different commercial sources (Lonza andPromocell), according to methods described in Example 1. The angiogenicactivity was assessed using the in vitro endothelial tube formationassay described in Example 2. Briefly, the exosomes (2×10⁸ particles/50μl) were incubated with human umbilical cord vascular endothelial(HUVEC) cells for 12-16 hours on low growth factor Matrigel using aμ-well slide (Ibidi, Verona, Wis.). Tube length was assessed by imagecapture and analyzed using Angiogenesis Analyzer in ImageJ(http://rsb.info.nih gov/ij/) image processing program. Total tubelength formed per image/μ-well was calculated relative to total tubelength formed for HUVEC in complete EGM-MV2 medium (CM) containingangiogenic growth factors (VEGF and FGF2) and fetal bovine serum (FBS).

Exosomes derived from the 30-MV2-6 cell line were compared to earlypassage BM-MSCs from the two different sources, Promocell (Heidelberg,Germany) (FIG. 4, panel A) and from Lonza (Basel, Switzerland) (FIG. 4,panel B). In both cases the angiogenic activity of 30-MV2-6 derivedexosomes was greater than the angiogenic activity of BM-MSC derivedexosomes. The total tube length of HUVECs incubated in basal medium with30-MV2-6 derived exosomes (in PBS) was similar to HUVECs incubated incomplete EGM-MV2 medium and significantly greater than BM-MSC derivedexosomes (from either source) or HUVEC incubated in basal EGM-MV2 mediumand PBS alone. The total tube length resulting from BM-MSC derivedexosomes was on average slightly higher than PBS in basal medium (BM)but the difference was not statistically significant (p<0.05). Theseresults indicate that embryonic endothelial progenitor stem cell linederived exosomes are advantageous over adult BM-MSC derived exosomes forinducing angiogenic activity in endothelial cells.

BM-MSC and 30-MV2-6 derived exosomes were further compared in a doseresponse experiment to determine differences in their potency. Exosomeswere prepared and their concentration determined using nanoparticletracking analysis (NTA; Nanosight, Malvern Instruments Ltd, Malvern,Worcestershire, UK). ELISA was used to confirm the presence oftranspanins CD63, CD81 and CD9 that are typically expressed on exosomes.The 30-MV2-6 exosomes were tested at doses ranging from 50 million to400 million exosomes per well. The BM-MSC exosomes were tested at dosesranging from 400 to 1200 million exosomes per well, because nosignificant activity was observed at 200 million exosomes per well. Theresults, shown in FIG. 5 indicate that the angiogenic response of HUVECcells to 30-MV2-6 exosomes is dose responsive starting at 50 million perwell and saturating at doses of >200 million exosomes per well (FIG. 5,panel A). In contrast, the BM-MSC exosomes showed a dose response ofincreasing angiogenic activity at doses from 400 million to 1200 millionexosomes per well (FIG. 5, panel B). The potency of 30-MV2-6 derivedexosomes was at least 6-fold greater than that of BM-MSC derivedexosomes, having the equivalent activity at 200 million exosomes perwell as BM-MSC derived exosomes at 1200 million exosomes per well (FIG.5, panel B).

Example 4 Comparison of miRNA Content in Exosomes Derived from a HumanEmbryonic Progenitor Cell Line versus Exosomes Derived from AdultBM-MSCs

The miRNA content of the 30-MV2-6 derived exosomes was analyzed andcompared to the miRNA content of the less angiogenic BM-MSC exosomes.RNA was extracted and purified from exosomes using the miRNeasy mini kitaccording to the manufacturer's recommended protocol (Qiagen, Hilden,Germany). The exosome RNA was quantified using a Nanodropspectrophotometer and cDNA was prepared from the RNA using the miScriptII RT kit (Qiagen, Hilden, Germany) according to the manufacturer'srecommended protocol. The exosome cDNA was amplified by polymerase chainreaction (PCR) using the miScript pre-AMP PCR kit (Qiagen) and miScriptpre-AMP pathway primer mix (human miFinder MBHS-001Z; Qiagen) accordingto the manufacturer's recommended protocol. Relative miRNA levels wereassessed for 84 human miRNAs by quantitative PCR using the humanmiFinder miScript miRNA PCR array (#331221; Qiagen) according to themanufacturer's recommended protocol. The results were analyzed using theAACT method of relative quantitation available at(http://perdataanalysis.sabiosciences.com/mima).

There were substantial differences in the miRNA content of the 30-MV2-6derived exosomes and BM-MSC derived exsomes. The miRNAs with greaterthan 6-fold difference between 30-MV2-6 exosomes and BM-MSC exosomes areshown in FIG. 6A scatter plot. Table 2 lists miRNAs that are more than2-fold overexpressed in 30-MV2-6 exosomes relative to BM-MSC exosomesand Table 3 lists miRNAs that are more than 2-fold underexpressed in30-MV2-6 exosomes relative to BM-MSC exosomes.

TABLE 2 miRNAs overexpressed in 30-MV2-6 exosomes compared to BM-MSCexosomes Fold miRNA Mature ID Difference hsa-miR-155-5p 3.98hsa-miR-18a-5p 2.54 hsa-miR-374a-5p 2.69 hsa-miR-126-3p 77.60

TABLE 3 miRNAs underexpressed in 30-MV2-6 exosomes compared to BM-MSCexosomes Fold miRNA Mature ID Difference hsa-miR-142-5p −56.29hsa-miR-9-5p −14.69 hsa-miR-27b-3p −6.92 hsa-miR-101-3p −4.82hsa-let-7d-5p −3.12 hsa-miR-16-5p −10.56 hsa-let-7g-5p −7.60hsa-miR-30c-5p −3.26 hsa-miR-96-5p −11.14 hsa-miR-185-5p −3.44hsa-miR-142-3p −17.38 hsa-miR-24-3p −9.55 hsa-miR-181b-5p −2.03hsa-miR-302b-3p −42.52 hsa-miR-30b-5p −3.34 hsa-miR-21-5p −16.16hsa-miR-15b-5p −4.08 hsa-miR-223-3p −18.36 hsa-miR-194-5p −2.69hsa-miR-15a-5p −4.25 hsa-miR-125b-5p −38.36 hsa-miR-99a-5p −10.43hsa-miR-29b-3p −15.10 hsa-miR-29a-3p −35.08 hsa-miR-141-3p −4.30hsa-let-7a-5p −4.03 hsa-miR-124-3p −13.56 hsa-miR-92a-3p −2.28hsa-miR-23a-3p −5.74 hsa-miR-25-3p −3.16 hsa-let-7e-5p −2.35hsa-miR-376c-3p −752.81 hsa-miR-144-3p −57.26 hsa-miR-195-5p −10.03hsa-miR-143-3p −82.38 hsa-miR-191-5p −4.88 hsa-let-7i-5p −9.62hsa-miR-302a-3p −17.39 hsa-miR-222-3p −2.31 hsa-let-7b-5p −35.56hsa-miR-186-5p −3.40 hsa-miR-196b-5p −71.33 hsa-miR-27a-3p −3.42hsa-miR-22-3p −4.58 hsa-miR-130a-3p −2.68 hsa-let-7c-5p −10.92hsa-miR-29c-3p −24.99 hsa-miR-140-3p −2.98 hsa-miR-128-3p −2.77hsa-let-7f-5p −2.89 hsa-miR-122-5p −9.20 hsa-miR-100-5p −10.00hsa-miR-302c-3p −144.85

The miRNA with the highest relative expression in 30-MV2-6 exosomescompared to BM-MSC exosomes is miR-126-3p (77.6-fold difference, Table2). MiR-126 is a known angiogenic miRNA (“angiomiR”) that is endothelialcell-specific and has been shown to regulate both vascular integrity anddevelopmental angiogenesis. Fish et al. (2008) Dev. Cell 15(2):272; Zouet al. (2011) Circ. Res. 108 (2):201; Jakob and Landmesser (2012)Cardiovasc. Res. 93(4):614; Nicoli et al. (2010) Nature 464(7292): 1196.Induction of miR-126 in endothelial cells and transport of miR-126 viaexosomes has been shown to be important for the effective treatment ofmyocardial infarction (MI) using transplanted cardiosphere derived cellsin a mouse model. Ong et al. (2014) Circulation 130 (11 Suppl 1):S60.

Accordingly, the clonal human embryonic progenitor cell line derived,miR-126-containing exosomes of the instant invention can be used in invitro angiogenesis studies as well as in treatment of myocardialinfarction and other ischemic conditions, either by themselves or incombination with transplanted cells.

RNA from 30-MV2-6 exosomes was also used to compare the miRNA content ofangiogenic versus non-angiogenic exosomes. Exosomes derived from HT1080cells (a human sarcoma cell line) are not angiogenic in the HUVEC invitro angiogenesis assay at 2.0×10⁸ exosomes, a dose at which 30-MV2-6exosomes show maximum angiogenic activity. HT1080 exosome RNA wasanalyzed on the miFinder miScript PCR array of 84 human miRNAs asdescribed above and compared to 30-MV2-6 and BM-MSC exosome RNA (Table4).

TABLE 4 Exosomal miRNAs in BM-MSC and 30-MV2-6 Relative to HT1080exosomes BM-MSC/ 30-MV2-6/ HT1080 HT1080 Fold Fold miRNA Mature IDDifference Difference hsa-miR-142-5p 13.3253 0.2367 hsa-miR-9-5p 19.94371.3579 hsa-miR-150-5p 4.3783 3.3457 hsa-miR-27b-3p 100.911 14.5862hsa-miR-101-3p 14.0298 2.9096 hsa-let-7d-5p 26.3588 8.441hsa-miR-103a-3p 4.5649 5.7031 hsa-miR-16-5p 38.0552 3.605 hsa-miR-26a-5p41.3044 24.3186 hsa-miR-32-5p 15.9192 14.3894 hsa-miR-26b-5p 52.267731.9112 hsa-let-7g-5p 76.0374 10.0027 hsa-miR-30c-5p 17.5335 5.3714hsa-miR-96-5p 1.1148 0.1 hsa-miR-185-5p 29.375 8.5466 hsa-miR-142-3p0.5468 0.0315 hsa-miR-24-3p 40.061 4.1927 hsa-miR-155-5p 1.0346 4.118hsa-miR-146a-5p 1.4501 1.0803 hsa-miR-425-5p 3.3003 2.8192hsa-miR-181b-5p 16.2276 8.0058 hsa-miR-302b-3p 45.0196 1.0589hsa-miR-30b-5p 19.9194 5.9589 hsa-miR-21-5p 52.4899 3.2475hsa-miR-30e-5p 4.4078 3.3143 hsa-miR-200c-3p 2.2547 1.3309hsa-miR-15b-5p 22.3822 5.4881 hsa-miR-223-3p 202.2804 11.0146hsa-miR-194-5p 8.5348 3.1718 hsa-miR-210-3p 1.8074 0.957 hsa-miR-15a-5p17.2702 4.0614 hsa-miR-181a-5p 28.6581 16.7792 hsa-miR-125b-5p 31.29420.8158 hsa-miR-99a-5p 13.0061 1.2466 hsa-miR-28-5p 11.1092 8.1182hsa-miR-320a 17.2154 13.7088 hsa-miR-125a-5p 13.0238 8.8833hsa-miR-29b-3p 9.9121 0.6563 hsa-miR-29a-3p 51.1831 1.4592hsa-miR-141-3p 7.9244 1.841 hsa-miR-19a-3p 4.5159 3.3341 hsa-miR-18a-5p7.7906 19.7947 hsa-miR-374a-5p 45.91 123.3183 hsa-miR-423-5p 20.469818.6725 hsa-let-7a-5p 25.3493 6.294 hsa-miR-124-3p 20.2226 1.4916hsa-miR-92a-3p 14.4072 6.3169 hsa-miR-23a-3p 52.5584 9.1492hsa-miR-25-3p 23.7331 7.5106 hsa-let-7e-5p 22.9181 9.7439hsa-miR-376c-3p 2411.7739 3.2037 hsa-miR-126-3p 123.9456 9618.223hsa-miR-144-3p 114.87 2.0061 hsa-miR-424-5p 57.34 92.8881 hsa-miR-30a-5p3.966 3.4279 hsa-miR-23b-3p 19.0703 13.0079 hsa-miR-151a-5p 12.860425.4071 hsa-miR-195-5p 40.2617 4.0149 hsa-miR-143-3p 273.1733 3.316hsa-miR-30d-5p 3.9436 4.0508 hsa-miR-191-5p 21.2256 4.3493 hsa-let-7i-5p62.9552 6.545 hsa-miR-302a-3p 37.209 2.14 hsa-miR-222-3p 22.8182 9.8699hsa-let-7b-5p 129.1842 3.6327 hsa-miR-19b-3p 5.758 3.3451 hsa-miR-17-5p4.6186 7.3351 hsa-miR-93-5p 16.2937 16.7172 hsa-miR-186-5p 14.80154.3526 hsa-miR-196b-5p 15.3129 0.2147 hsa-miR-27a-3p 48.4243 14.148hsa-miR-22-3p 13.8229 3.0157 hsa-miR-130a-3p 11.5632 4.3074hsa-let-7c-5p 93.5201 8.5615 hsa-miR-29c-3p 39.8245 1.5935hsa-miR-140-3p 9.8899 3.3169 hsa-miR-128-3p 21.7273 7.8472 hsa-let-7f-5p30.5392 10.5782 hsa-miR-122-5p 71.7561 7.8014 hsa-miR-20a-5p 4.61199.1362 hsa-miR-106b-5p 5.093 8.4218 hsa-miR-7-5p 4.8173 6.4945hsa-miR-100-5p 14.7509 1.4748 hsa-miR-302c-3p 120.5214 0.832

The data in Table 4 and FIG. 6B illustrate the dramatic differences inthe miRNA content of the three types of exosomes. These data show thatHT1080 exosomes contain the lowest amount of miRNA for all miRNAstested, except for miR-96-5p and miR-142-3p, which are lowest in30-MV2-6 exosomes. The miRNA miR-142-3p is expressed at highest levelsin HT1080 exosomes and is known to repress several inhibitors ofoncogenic transformation. It is mimicked by Kaposi sarcoma viral miRNA,miR-K10a. Forte et al. (2015) J Virol 89(4): 2333. The miR-96-5p miRNAis present in highest levels in BM-MSC exosomes and is higher in HT1080exosomes than 30-MV2-6 exosomes. This miRNA is thought to be involved inosteogenic and adipogenic differentiation in BM-MSCs (Laine et al.(2012) J Cell Biochem. 113(8):2687) but is also involved in tumor cellproliferation (Lin et al. (2010) Plos One 5(12):e15797; Haflidadottir etal. (2013) Plos One 8(8):e72400). Strikingly the miRNA with the highestlevels in 30-MV2-6 exosomes relative to HT1080 exosomes is miR-126-3p.This known angiogenic miRNA is present at a 9618-fold higher level in30-MV2-6 exosomes than HT1080 exosomes. MiR-155 is 4-fold higher in30-MV2-6 exosomes than BM-MSC or HT1080 exosomes and is anti-angiogenicbut pro-arteriogenic. Pankrtaz et al. (2015) Circulation 131(18):1575.Of the 9 remaining miRNAs that are highest in 30-MV2-6 exosomes, 6 areknown to be involved in angiogenesis. The miRNAs miR-18a-5p, miR-20a-5p,miR-424-5p, miR-17-5p, and miR-7-5p miRNAs have anti-angiogenicactivity. However, none of these anti-angiogenic miRNAs are more than2.5-fold enriched in 30-MV2-6 exosomes compared to BM-MSC exosomes. Thepro-angiogenic miR-106b is 9-fold enriched in 30-MV2-6 but only 4-foldenriched in BM-MSC exosomes compared to HT1080 exosomes. It is neededfor neovascularization after hind limb ischemia. Semo et al. (2014) EurHeart J. 35(45):3212.

Notably, several anti-angiogenic miRNAs, including miR-143-3p (Climentet al. (2015) Circ Res. 116(11):1753), miR-223-3p (Dai et al. (2014)Plos One 9(10):e108468), miR-222-3p (Suarez and Sessa (2009) Circ Res.104(4):442), miR-15a, miR-15b and miR-16 (Spinetti et al. (2013) CircRes. 112(2):335; Liu et al. (2012) Cell Physiol Biochem. 29(5-6):851))were enriched for and/or present at highest level in the BM-MSC-derivedexosomes.

Example 5 Comparison of Angiogenic Activity of Exosomes Derived fromVarious Clonal Embryonic Stem Cell Lines and Exosomes Derived from theParental Pluriopotent Stem Cell Lines

Clonal embryonic progenitor cell lines were prevously established fromhuman pluripotent stem (hPS) cell lines using methods previouslydescribed. West et al. (2008) Regen Med. 3(3):287. The resulting celllines are not immortalized but have higher replicative potential thanprimary cell lines because of their long telomere length that is nearthat of the parental hPS cell line from which they are derived. A widediversity of cell types was produced by exposing hPS cells to an arrayof cell culture medium, cell matrix, and growth conditions followed byselective pressure for clonal growth and scalability. Over 140 such celltypes have been determined to be distinct by analysis of totaltranscribed RNA using standard Illumina microarrays. The in vitroangiogenesis assay (described in detail in Example 2) was used to screenclonal embryonic progenitor cells for production of angiogenic exosomes.As shown in Table 5, most embryonic endothelial progenitor cell-derivedexosomes have angiogenic activity in the range of 30-MV2-6 derivedexosomes (+; relative tube length (RTL) >0.75 and <1.25). The 30-MV2-9exosomes scored highest (++; RTL >1.25). Two endothelial progenitorlines scored negative (-; RTL <0.75). Exosomes from an osteochondralline, primary fibroblasts (BJ), BM-MSCs, and a human sarcoma cell line(HT1080) were also negative. The two clonal smooth muscle cellprogenitor cell lines and one clonal pericyte line tested were positivein the in vitro vascular tube formation assay. Exosomes prepared fromconditioned medium of the parental human embryonic stem cell lines H9(WA09) and ESI-017 were also positive in the in vitro vascular tubeformation assay.

TABLE 5 TABLE 5: Angiogenic activity of PureStem vascular progenitorsand other cell lines. Relative tube Angiogenic Cell Line Source CellType length Index 30-MV2-9 PureStem Endothelial 1.67 ++ 30-MV2-6PureStem Endothelial 1.07 + 30-MV2-7 PureStem Endothelial 0.94 +30-MV2-17 PureStem Endothelial 0.76 + 30-MV2-19 PureStem Endothelial0.84 + RP1-MV2-8 PureStem Endothelial 1.07 + 30-MV2-14 PureStemEndothelial 1.00 + 30-MV2-15 PureStem Endothelial 1.00 + RP1-MV2-8PureStem Endothelial 1.07 + 30-MV2-24 PureStem Endothelial 0.41 −30-MV2-4 PureStem Endothelial 0.72 − W10 PureStem Smooth Muscle 0.83 +Z11 PureStem Smooth Muscle 0.92 + E164 PureStem Pericyte 0.86 + PC-M hESPericyte 0.47 − BJ Primary Foreskin 0.61 − Fibroblast SM30 PureStemMSC-like 0.71 − MSCs Primary Adult 0.68 − BM-MSC HT1080 TransformedSarcoma 0.71 − H9 Embryonic Pluripotent 1.06 + ESI-017 EmbryonicPluripotent 1.27 +

Example 6 In Vivo Angiogenic Activity of Embryonic Progenitor Stem CellDerived Exosomes

Angiogenic activity of exosomes was assessed in vivo using the Matrigelplug assay in mice as previously described. Sahoo et al. (2011) CircRes. 109(7):724. Immunocompromised mice (Female Nu/J mice aged 6-8weeks; 2 plugs/mouse; 2 mice/group) were injected subcutaneously withapproximately 300 μl of Matrigel containing PBS, 4×10⁸/ml exosomes, or150 ng/ml bFGF plus 60 ng/ml VEGF (positive control). The plugs wereremoved at day 14 after implant followed by fixation and paraffinembedding. The sections were stained with hematoxylin and Eosin (H&E)for histological examination and stained with von Willabrand factorantibody for detection of endothelial cells.

The data indicate that 30-MV2-6 exosomes are angiogenic in the Matrigelplug assay (FIG. 7). The exosome containing plugs show regions ofinfiltration of cells into the plug with vessel formation (FIG. 7,panels A and C). The positive control plugs containing growth factorshave regions of vessel formation (not shown) Immunostaining withantibody against von Willabrand factor (FIG. 7, panels B and D)confirmed the endothelial identity of cells lining the vessel structuresobserved by H&E staining. The PBS control plugs show less cellinfiltration and no vessel formation (FIG. 7, panel E).

Example 7 Scale-Up of Clonal Embryonic Progenitor Stem Cells for ExosomeProduction

Clonal embryonic progenitor cell lines described here are advantageousover other sources of biologically active exosomes because of theirscalability. The parental pluripotent stem cell line to 30-MV2-6 whichalso produces angiogenic exosomes is costly to scale up because of therequirements for specialized medium and cell matrix (e.g. Matrigel).Primary endothelial stem cells or mesenchymal stromal cells rapidly losedifferentiation and proliferative capacity upon culture in vitro.Typically MSCs begin to senesce in culture after 7-12 passages(approximately 10 population doublings) and show multiple changesincluding altered surface marker expression and increasedautofluorescence. Wagner et al. (2008) Plos One 3(5):e2213. In contrast,human embryonic clonal progenitor lines such as the cell lines of theinstant invention are grown under standard tissue culture conditions andmedium and are highly scalable with typical replicative lifespans of 60to 100 population doublings.

The Terumo Quantum Cell Expansion system (the bioreactor used in theinstant example) is an automated hollow fiber cell culture platformdesigned for GMP compatible production of cells for use in cell therapy.The bioreactor was seeded at a density of approximately 900 cells/cm²with approximately 4.0×10⁷ 30-MV2-6 cells (passage 9) and the cells werecultured for 13 days under their standard growth conditions of EGM-MV2medium and 5% oxygen. The exosomes were collected by exchanging thecomplete medium for conditioning medium (basal EGM-MV2 medium withoutserum added; alternatively PBS may be also used). The conditioningmedium was left in the bioreactor for 16 hours and collected for exosomepurification. The cells were harvested by exchanging medium with a 0.25%trypsin solution to remove cells for the reactor, tested for viabilityand counted. Cells were scaled over 10-fold from the initial 40 millionto approximately 440 million. The purified exosomes were quantifiedusing CD63 detection ELISA (alternatively, nanoparticle trackinganalysis as described in Example 1 may be used to quantify theexosomes). The yield of exosomes from one bioreactor run is at least2.3×10¹⁰, which is equivalent to the approximate exosome yield from 72T-225 flasks of 30-MV2-6 cells.

The purified exosomes were tested for angiogenic activity at a dose of2.0×10⁶ exosomes per well in the in vitro tube formation assay(described in detail in Example 2). As shown in FIG. 8, the angiogenicactivity of exosomes prepared from media conditioned by 30-MV2-6 cellsgrown in T-flasks was equivalent to the angiogenic activity of exosomesprepared from medium conditioned by 30-MV2-6 cells grown in the QuantumCell Expansion system.

Example 8 Effect of Oxygen Concentration and Conditioning Medium onExosome Activity

Hypoxia has been reported to increase exosome production from mammaliancells (Tadokoro et al. (2013) J Biol Chem. 288(48):34343; King et al.(2012) BMC Cancer 12:241). Furthermore, clonal embryonic progenitor celllines are derived and maintained under low oxygen (5%). West et al.(2008) Regen Med. 3(3): 287. Therefore, 1% oxygen was tested for exosomeproduction to determine if increasing hypoxia will increase exosomeproduction or angiogenic activity.

Other stress conditions can also have an effect on exosome yield oractivity. Serum starvation is used to induce exosome production.Nutrient deprivation was tested by using PBS as the conditioning medium.The use of PBS versus basal EGM-MV2 medium for conditioning the cellswas also tested.

The results shown in FIG. 9 indicate that there is no significantdifference in angiogenic activity of isolated exosomes when the mediumwas conditioned by cells incubated in 1% or 5% oxygen. These data alsoindicate the exosome angiogenic activity is not significantly differentwhen PBS is used as the conditioning media compared to when the basalmedium is used as the conditioning medium, although there is a trendtoward higher activity when PBS is used.

Example 9 Quantitation of Exosome Concentration by ELISA Detection ofCD63 on Intact Exosomes

There is a need for simple and convenient method to measure theconcentration of exosome particles in a purified preparation ofexosomes. Currently available ELISA kits for measuring exosomeconcentration (System Biosciences, Inc., Mountain View, Calif.) requirelysing exosomes and have a lower limit of detection of approximately2.0×10⁸ exosomes. It is advantageous to measure low concentrationsamples directly without diluting the sample in a lysis buffer.Moreover, lysing the exosomes releases other proteins and nucleic acidsthat potentially interfere with the assay. The method described hereintakes advantage of markers commonly presented on the surface ofexosomes, such as transpanins CD63, CD9 and CD81 and allows forquantitation of intact exosomes.

A standard curve is prepared from exosome samples of known concentration(ranging from 5×10⁸ to 8×10⁷ exosomes/mL). The unknown samples areprepared in PBS or a buffer exchanged into PBS. Samples of intactexosomes are bound to 96 well ELISA plate wells in PBS at 50 μl/well forat least 16 hours at 37° C. The wells are washed 3×5 minutes in washbuffer (e.g. TBS-Tween). The wells are incubated with a mouse monoclonalantibody prepared in a suitable blocking buffer (e.g. PBS containingexosome depleted FBS and 0.05% Tween 20) that recognizes theextracellular domain of CD63 on intact exosomes for 1 hour at roomtemperature. The wells are washed again 3×5 minutes at room temperature.The wells are incubated with a suitable secondary antibody in a blockingbuffer for detection of mouse anti-CD63 antibody bound to exosomes onthe plate surface (e.g. HRP conjugated goat anti-mouse IgG) for 1 hourat room temperature. The wells are washed again 3×5 minutes at roomtemperature and the wells incubated with 50 tl of HRP substrate (e.g.Supersensitive TMB ELISA substrate) for 30 minutes at room temperature.The wells are washed 3×5 minutes at room temperature and 50 μl of stopbuffer (0.16M sulfuric acid) is added to provide a fixed endpoint. Theconcentration of exosomes is quantitated by measuring the absorbance ofeach well at 450 nm.

An example of a standard curve and quantitation of samples is shown inFIG. 10.

1. An exosome isolated from a progenitor cell line.
 2. The exosome ofclaim 1, wherein the progenitor cell line is a human progenitor cellline.
 3. The exosome of claim 1, wherein the progenitor cell line is aclonal progenitor cell line.
 4. The exosome of claim 1, wherein theprogenitor cell line is an endothelial progenitor cell line.
 5. Theexosome of claim 1, wherein the exosome contains CD63.
 6. The exosome ofclaim 1, wherein the exosome contains one or more miRNAs listed in Table2 or Table
 4. 7. The exosome of claim 1, wherein the exosome containsone or more angiogenic miRNAs.
 8. The exosome of claim 1, wherein theexosome contains miR-126.
 9. The exosome of claim 1, wherein theprogenitor cell line expresses CD31 and CD34.
 10. The exosome of claim1, wherein the progenitor cell line expresses one or more genes listedin Table1.
 11. The exosome of claim 1, wherein the progenitor cell lineis 30-MV2-6.
 12. The exosome of claim 1, wherein the exosome enhancesthe formation of vascular tubc liko tube-like formations when contactedwith an endothelial cell.
 13. The exosome of claim 1 further comprisinga pharmaceutical carrier.
 14. A method of isolating an exosome from aclonal progenitor cell comprising: 1) culturing the clonal progenitorcell in a suitable medium or buffer for a time sufficient to allow theclonal progenitor cell to exocytose exosomes into the culture medium orbuffer; 2) harvesting the medium or buffer from the cell culture of step1; and 3) isolating the exosomes from the medium or buffer of step 2,thereby isolating an exosome from a clonal progenitor cell. 15-16.(canceled)
 17. The method of claim 14, wherein after step 2, aprecipitating agent is added to the medium or buffer.
 18. The method ofclaim 17, wherein the precipitating agent comprises polyethylene glycol.19-20. (canceled)
 21. The method of claim 14, wherein after step 2, themethod further comprises a step of incubating the harvested medium orbuffer. 22-35. (canceled)
 36. A method of inducing or enhancing a cell'sability to form vascular tube-like structures comprising contacting acell capable of making vascular tube-like structures with an exosomeisolated from a progenitor cell thereby inducing or enhancing a cell'sability to form vascular tube-like structures.
 37. The method of claim36, wherein the cell capable of making vascular tube-like structures isan endothelial cell.
 38. The method of claim 37, wherein the endothelialcell is a HUVEC. 39-53. (canceled)